Supercriticial fluid extraction of flavors and fragrances from Hyssopus officinalis L. cultivated in Iran

Hyssopus officinalis L. (hyssop) as a food ingredient has its own importance in flavor industry and also in sauce formulations. Supercritical fluid extraction (SFE) of hyssop, cultivated in Iran, was performed at various pressures, temperatures, extraction (dynamic and static) times and modifier (methanol) concentrations using an orthogonal array design with an OA₂₅(5⁵) matrix conditions. Pressure, temperature and modifier in the SFE system influenced the extraction yield. Also, the composition of the extracted oils was greatly impacted by the operating conditions. Main components of the extracts under different SFE conditions were sabinene (4.2–17.1%, w/w), iso-pinocamphene (0.9–16.5%) and pinocamphene (0.7–13.6%). The extraction of sabinene, for example, was favored at 100 atm, 55 °C, 1.5% (v/v) methanol, 30 min dynamic time and 35 min static time. Use of SFE under different conditions can allow targeting the extraction of different constituents.     

Supercritical fluid extraction of forskolin from Coleus forskohlii roots

Forskolin (FSK), a labdane diterpene compound having high nutraceutical and therapeutic activity has been extracted from dried Coleus forskohlii roots using supercritical carbon dioxide (SC-CO₂). The solubility parameter of FSK, CO₂, and entrainer solvents was calculated and validated with experimental results. Theoretically, pressure and temperature had significant effect on extraction of FSK. A maximum of 50.32% recovery of FSK was obtained after SC-CO₂ extraction at 40 °C, 250 bar and extraction time of 60 min. Use of methanol as an entrainer at 20% v/w of dried C. forskohlii roots under optimized conditions improved the recovery of FSK to 74.29%. The recovery of FSK further increased marginally to 77.08% when pre-treated with ultrasonication and commercial enzyme preparation of Stargen® 002 and Accellerase® 1500.     

Development of a solid-state fermentation process for production of an alpha amylase with potentially interesting properties

Ca-independency with potential activity and stability at low pH are among the most interesting characteristics of α-amylase in starch industry. In this attempt the synergetic effect of low pH on activity of crude Ca-independent α-amylase isolated from a native Bacillus sp. KR-8104 in solid-state fermentation (SSF) was studied using wheat bran (WB) as a substrate. The effects of different parameters including moisturizing agents, solid substrate to moisture ratio, particle size, incubation temperature and period, inoculum (v/w) and supplementation with 1% (w/w) different carbon and nitrogen sources on enzyme production were investigated. Maximum enzyme production of 140 U/g dry fermented substrate was obtained from wheat bran moistened with tap water at a ratio of 1:1.5 and supplemented with 1% (w/w) NH₄NO₃ and 1% (w/w) lactose after 48 h incubation at 37 °C. Even though the production of α-amylase was lower at 40 and 45 °C, the viable cell count was higher. In addition response surface methodology (RSM) was applied to find optimum conditions of temperature and pH on crude amylase activity. Using central composite design (CCD) a quadratic mathematical model equation was derived for the prediction of enzyme activity. The results showed that the model was in good agreement with experimental results, with R² = 0.90 (p < 0.0001) and the low pH has a synergetic effect on enzyme activity at higher temperature.     

Supercritical CO2 cell breaking extraction of Lycium barbarum seed oil and determination of its chemical composition by HPLC/APCI/MS and antioxidant activity

The extraction parameters for oil extraction from Lycium barbarum seed including extraction pressure, temperature and time were optimized using an orthogonal test design. The optimum conditions for supercritical CO₂ extraction were as follows: extraction pressure, 30 MPa; extraction temperature, 45 °C; dynamic extraction time, 60 min; CO₂ flow, 25 kg/h. The oil yield under the conditions proposed was 19.28 g/100 g. The effect of cell wall breakage pretreatment was investigated by supercritical CO₂ rapid depressurization, and results indicated this pretreatment could result in a rapid and efficient extraction. A sensitive fluorescent reagent 2-(11H-benzo[a]carbazol-11-yl) ethyl 4-methylbenzenesulfonate (BCETS) was utilized as pre-column labeling regent to determine fatty acids (FA) from Lycium barbarum seed oils obtained by different extraction methods. The main FA were: C18:2, C18:1, C16, C20:6, C18:3, and C20. The oil from L. barbarum seed exhibited excellent antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl（DPPH）radical scavenging assay and β-carotene bleaching test, and its antioxidant activity compared well with the references ascorbic acid and α- tocopherol.     

Extraction of α-tocopherol and γ-oryzanol from rice bran

The aim of this research was to study the effect of operating mode (continuous versus batch+continuous), temperature, pressure and solvent on α-tocopherol and γ-oryzanol extraction from rice bran (Oryza sativa Linn.) and compare the efficiency of three extraction methods: SC-CO₂ extraction, solvent extraction and soxhlet extraction. Three sets of experiments were performed. First, extraction using SC-CO₂ was performed over a range of temperatures and pressures (45-65 °C and at 38 and 48 MPa), and at a CO₂ flow rate of 0.45 mL/min. The results showed that the best conditions for α-tocopherol extraction were 55 °C, 48 MPa in the batch+continuous mode. For γ-oryzanol, the best conditions were 65 °C, 48 MPa and in the continuous mode. In the second set of experiments, solvent extraction using hexane and ethanol at 32 and 55-60 °C was studied. The results showed that none of the solvents could extract α-tocopherol; however, ethanol at 55-60 °C was suitable for γ-oryzanol extraction. Finally, soxhlet extraction experiments using hexane for α-tocopherol and ethanol for γ-oryzanol were also performed. In summary, SC-CO₂ was found to be the best solvent for extracting both α-tocopherol and γ-oryzanol from rice bran, because of its higher yields and extraction rate.     

Antioxidant activity of microwave-assisted extract of Buddleia officinalis and its major active component

Buddleia officinalis Maxim, commonly used as rice dye for festivals, was extracted with ethanol using microwave-assisted extraction and Soxhlet extraction. The antioxidant activities of microwave-assisted extract of B. officialis (MEB) and Soxhlet extract of B. officianils (SEB) at the optimum extraction conditions were evaluated and compared with synthetic antioxidant butylated hydroxytoluene (BHT) employing DPPH free radical assay, ABTS assay, total antioxidant activity and reducing power. MEB and SEB had stronger antioxidant activities than BHT in all assays except reducing power, and the effects decreased as follows: MEB > SEB > BHT. The total phenolic contents of MEB and SEB reached 113.56 mg/g and 100.94 mg/g dry weight of extract, respectively, expressed as pyrocatechol equivalents, while the total flavonoids contents were 75.33 mg/g and 62.56 mg/g dry weight of extract, respectively, expressed as catechin equivalents (P < 0.05). Higher phenolic and flavonoids compounds may be major contributors to their higher antioxidant activities. Following activity-oriented separation, luteolin was isolated as an active principle, which exhibited excellent free radical scavenging activities with DPPH IC₅₀ 3.09 μg/ml and ABTS IC₅₀ 2.20 μg/ml.     

Crinumin,a chymotrypsin-like but glycosylated serine protease from Crinum asiaticum: Purification and physicochemical characterisation

Plant latex could be a potential source of novel proteases usable in the food and feed industries because of broad substrate specificity with high stability in extreme conditions. Crinumin, a glycosylated serine protease with chymotrypsin-like activity was purified from the latex of Crinumasiaticum using cation-exchange column chromatography. Crinumin shows activity over a wide range of pH (4.5–11.5 and optimum at 8.5), temperature (75 °C and optimum at 70 °C) and is also functional against chaotrophs, organic solvents, and detergents, even after prolonged exposure. The molecular mass (67.7 kDa), extinction coefficient (17.7), isoelectric point (6.9), and numbers of tryptophan (13), tyrosine (24) and cysteine (15 with 7 disulphide bridges) residues were estimated. K_m of the enzyme was 31.7 μM with casein and 5 × 10⁴ μM with N-succinyl-l-phenylalanine-p-nitroanilide. Easy availability of the aqueous latex, simple purification procedure, high yield (33%), stability and activity in adverse conditions makes it applicable for the pharmaceutical and food industries.     

Response surface optimisation for the extraction of phenolic compounds and antioxidant capacities of underutilised Mangifera pajang Kosterm. peels

The optimum extraction conditions for highest recovery of total phenolics content (TPC) and antioxidant capacities (AC) were analysed for Mangifera pajang peels (MPP), using response surface methodology. The effects of ethanol concentration (X₁: 20–80%), extraction temperature (X₂: 30–65 °C) and liquid-to-solid ratio (X₃: 20–50 mL/g) on the recovery of total phenolics (Y₁) and antioxidant capacity (Y₂) were investigated. A second order polynomial model produced a satisfactory fitting of the experimental data with regard to total phenolic content (R² = 0.9966, p < 0.0001) and antioxidant capacity (R² = 0.9953, p < 0.0001). The optimum extraction conditions for TPC were 68%, 55 °C and 32.7 mL/g, and for AC were 68%, 56 °C and 31.8 mL/g, respectively. Predicted values for extraction of TPC and AC agreed well with the experimental values. Liquid chromatography–mass spectrometry of the optimally obtained extracts from MPP revealed the major phytochemicals as mangiferin, gallic acid, catechin and epicatechin.     

Extraction optimization of watermelon seed protein using response surface methodology

Extraction conditions for maximum protein recovery from watermelon (Citrullus vulgaris Cv Sugar baby) seed meal were investigated using response surface methodology. A central composite design with four independent variables: temperature (40, 45, 50, 55 and 60 °C); NaOH concentration (0.03, 0.06, 0.09, 0.12 and 0.15 g/L); extraction time (5, 10, 15, 20 and 25 min) and solvent/meal ratio (30:1, 40:1, 50:1, 60:1 and 70:1, v/w) was used to study the response variable (protein yield). The experimental values of protein yield ranged between 72.03 and 81.52 g/100 g seed meal. A second-degree equation for independent and response variables was computed and used to create the contour plots graphs. The predicted values obtained for protein yield revealed that coefficient of determination and standard error was 0.80 and 0.906, respectively. Optimum protein extraction was obtained with 0.12 g/L alkali concentration, 15 min extraction time and 70:1 (v/w) solvent/meal ratio at 50 °C. Confirmatory studies revealed that the protein yield under optimum conditions was 80.71 g/100 g seed meal.     

Extraction optimisation,purification and major antioxidant component of red pigments extracted from Camellia japonica

The optimised extraction conditions of red pigments (RP) from Camellia japonica obtained with an orthogonal design L₉(3⁴) were solid/liquid ratio, temperature, pH and extraction time as 1/10, 60 °C, 1.5 and 4 h, respectively. The RP were then purified by the macroporous resin method, which showed the resin LX-68 was appropriate for purifying the pigments from C. japonica. The antioxidant activities of these pigments were also investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical in vitro model systems. The DPPH scavenging activity of pigment extract was comparable to that of standard butylated hydroxyanisole (BHA). The IC₅₀ values of the RP and BHA were 4.55 and 4.17 μg ml⁻¹, respectively. The pigments showed higher hydroxyl radical-scavenging activities than that of mannitol at the same concentration. Following activity-oriented separation, (−)-epicatechin was isolated as an active principle, which exhibited excellent DPPH free radical scavenging activities with IC₅₀ 5.08 μg ml⁻¹.     

Determination of herbicide ZJ0273 residue in rapeseed by radioisotopic tracing method

Residue of herbicide ZJ0273 (propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate) in rapeseed under simulated field conditions was evaluated by radioisotopic tracing method. Radio-HPLC and HPLC–MS analysis revealed that the parent ZJ0273 is the only radioactive residue in rapeseed. The residue of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)[phenyl-U-¹⁴C₆]benzylamino)benzoate (RCZJ) in postharvest rapeseed from the rape seedlings (Brassica napus L.) in red clayey soil (S₁) and in fluvio marine yellow loamy soil (S₂) were 0.67 and 1.24 nmol/plant, respectively, based on the ¹⁴C radioactivity and the specific activity of the labelled ZJ0273. The RCZJs in the rapeseed (1 kg) from the seedlings in S₁ and in S₂ were 40.13 and 45.33 nmol, respectively, and the amounts of ZJ0273 in the rapeseed (1 kg) were 16.97 and 19.17 μg. The results suggested that only a trace level of residue of ZJ0273 presents in rapeseed due to the poor root absorption of ZJ0273 and the weak translocation of ZJ0273 from rape leaves.     

Extraction, thermal stability and kinetic behavior of pectinmethylesterase from hawthorn (Crataegus pubescens) fruit

Pectinmethylesterase (PME) extracted from hawthorn (Crataegus pubescens) fruit was evaluated for its thermal stability and kinetic behavior. The enzyme extraction process was established after studying different NaCl concentrations (0.5-3.0 moles/L). A maximum PME extraction of 51.61 units/mg protein was obtained using 2 moles/L NaCl. Kinetic parameters (K_m and V_max) were determined using a commercial citrus pectin and C. pubescens pectin as substrates. The effects of NaCl concentration, pH and temperature on PME activity were investigated. PME showed higher affinity for C. pubescens pectin (K_m and V_max of 2.84 mg/mL protein, and 64.10 units/mg protein, respectively) than for citrus pectin. C. pubescens PME extract showed maximum activity at 0.4 moles/L NaCl, pH 7.5, and 55 °C. The E_a and Q₁₀ for thermal activation were 36.27 kJ/mol and 2.01 (20-30 °C), respectively. About 50% of the activity still remained after heating for 25 min at 60 °C, and it was completely inactivated by incubation at 80 °C for 10 min. The Q₁₀ and E_a values for thermal inactivation reaction were 20.06 (70-80 °C) and 146.16 kJ/mol, respectively. These results provide useful information about the factors that affect the activity of C. pubescens PME, and might be used as a starting point for texture control during post-harvest handling and processing of this fruit.     

Supercritical carbon dioxide fluid extraction of Hibiscus cannabinus L. seed oil: A potential solvent-free and high antioxidative edible oil

The supercritical fluid extraction (SFE) trends and antioxidant activities of Hibiscus cannabinus seed oils were studied. SFE results indicate that extraction pressure is the major factor determining the oil yield. In comparison, classic Soxhlet extraction (SOX/L) yielded higher oil content than SFE (P < 0.05). However, no significant differences in oil content were observed in SFE at 600 bars/80 °C, rapid Soxhlet extraction (SOX/S) and conventional ultra-sonic assisted solvent extraction (SONIC) (P > 0.05). Antioxidant activities of H. cannabinus seed oils were compared with 7 types of commercial edible oils. DPPH scavenging activity test indicated that H. cannabinus seed oil extracted by SFE at 200 bars/80 °C possessed the highest antiradical activity whereas beta-carotene bleaching (BCB) assay revealed that all H. cannabinus seed oils (except for SFE at 400 bars/80 °C and 600 bars/80 °C) exhibited higher antioxidant activity than all commercial edible oils (P < 0.05). Thus, SFE – H. cannabinus seed oil may serve as an excellent source of solvent-free edible oil with high antioxidant properties.     

Proximate composition and extraction of carotenoids and lipids from Brazilian redspotted shrimp waste (Farfantepenaeus paulensis)

The chemical composition of redspotted shrimp (Penaeus paulensis) waste was investigated. The shrimp waste (freeze-dried heads, shells and tails) was found to have high protein (49% d.w.) and ash (27% d.w.) contents, but a low lipid content (4.9% d.w.) although the latter was higher than those found in other kinds of shrimp captured in Brazil. The fatty acid compositions showed that the lipids had a high content of unsaturated fatty acids, mainly EPA (C20:5; n-3) and DHA (C22:6; n-3). In order to establish an efficient and environmentally friendly recovery process for the astaxanthin (principal carotenoid and antioxidant present in the waste), the following processes were examined: traditional solvent extraction (TSE), super-critical fluid extraction (SC-CO₂) and super-critical fluid extraction with co-solvent (SC-CO₂ + ethanol). The temperature and pressure conditions for all the SC-CO₂ extractions were 50 °C and 30.0 MPa. The results showed that the mixture of 60% (v/v) n-hexane:isopropyl alcohol gave the highest (53 mg/kg waste) carotenoid extraction yield as compared to acetone, SC-CO₂ and SC-CO₂ + ethanol. The SC-CO₂ showed the lowest extraction yield of astaxanthin, but the addition of the entrainer (10% w/w) produced an important effect, increasing the astaxanthin extraction to values of 57.9%, similar to extraction with acetone (63.3%).     

Hollow fibre-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of flavonoids in Echinophora platyloba DC. and Mentha piperita

A simple, inexpensive and efficient three phase hollow fibre liquid phase microextraction (HF-LPME) technique combined with HPLC was used for the simultaneous determination of flavonoids in Echinophora platyloba DC. and Mentha piperita. Different factors affecting the HF-LPME procedure were investigated and optimised. The optimised extraction conditions were as follows: 1-octanol as an organic solvent, pH_donor = 2, pH_acceptor = 9.75, stirring rate of 1000 rpm, extraction time of 80 min, without addition of salt. Under these conditions, the enrichment factors ranged between 146 and 311. The values of intra and inter-day relative standard deviations (RSD) were in the range of 3.18–6.00% and 7.25–11.00%, respectively. The limits of detection (LODs) ranged between 0.5 and 7.0 ng mL⁻¹. Among the investigated flavonoids quercetin was found in E. platyloba DC. and luteolin was found in M. piperita. Concentration of quercetin and luteolin was 0.015 and 0.025 mg g⁻¹ respectively.     

Nitrogen extractability and functional properties of defatted Erythrina variegata flour

Defatted Erythrina variegata flour was prepared from dehusked seed meal by hexane extraction of residual oil. The resulting flour had 403 g kg⁻¹ of protein as compared to 282 g kg⁻¹ in the whole seed-defatted meal. Nitrogen extractability of the defatted flour in water was not much influenced by the length of extraction period above 40 min, but an increased extraction was observed at a higher liquid to solid ratio up to a studied limit of 1:60; the optimal ratio was found to be 1:30. The minimum of 26.9% nitrogen was extracted in the pH range 3.0–4.0 and maximum of 94.8% at pH 12. Addition of sodium chloride (0.1 or 0.5 M) broadened the pH range of minimum nitrogen extractability and shifted it toward a lower pH region. At both concentrations of sodium chloride, a marked increase in nitrogen extractability, in the pH range 3.0–7.0, was observed. Precipitation of protein from an extract of pH 10.0 was maximum (85.3%) at pH 4.75. A higher buffer capacity of the flour was observed in the acidic medium (0.195 mmol HCl g⁻¹ flour) than in alkaline medium (0.093 mmol NaOH g⁻¹). Water absorption and oil absorption values for the whole E. variegata seed flour and the dehusked, defatted flour were 1.81, 1.43 and 1.02, 1.52 kg kg⁻¹, respectively.     

Ultrasound-assisted production of bioethanol by simultaneous saccharification and fermentation of corn meal

An ultrasound-assisted liquefaction as a pretreatment for bioethanol production by simultaneous saccharification and fermentation (SSF) of corn meal using Saccharomyces cerevisiae var. ellipsoideus yeast in a batch system was studied. Ultrasound pretreatment (at a frequency of 40 kHz) was performed at different sonication times and temperatures, before addition of liquefying enzyme. An optimal duration of the treatment of 5 min and sonication temperature of 60 °C were selected, taking into account glucose concentration after the liquefaction step. Under the optimum conditions an increase of glucose concentration of 6.82% over untreated control sample was achieved. Furthermore, the SSF process kinetics was assessed and determined, and the effect of ultrasound pretreatment on an increase of ethanol productivity was investigated. The obtained results indicated that the ultrasound pretreatment could increase the ethanol concentration by 11.15% (compared to the control sample) as well as other significant process parameters. In this case, the maximum ethanol concentration of 9.67% w/w (which corresponded to percentage of the theoretical ethanol yield of 88.96%) was achieved after 32 h of the SSF process. A comparison of scanning electron micrographs of the ultrasound-pretreated and untreated samples of corn meal suspensions showed that the ultrasound stimulated degradation of starch granules and release of glucose, and thereby accelerated the starch hydrolysis due to the cavitation and acoustic streaming caused by the ultrasonic action.     

A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.     

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.     

Degradation of aflatoxins by extrusion cooking: Effects on nutritional quality of extrudates

Extrusion of artificially contaminated food is reported to degrade aflatoxins to varying degrees depending on the extrusion conditions. This work sought to determine the (1) efficacy of extrusion cooking in destroying naturally contaminated peanuts and (2) nutritional quality of decontaminated peanut meal. Naturally contaminated peanut meal was extruded by varying the moisture (20, 28, 35 g/100 g), pH (7.5, 9.5) and extruder die diameter (2.5, 3, 3.5, 4.0 mm). Aflatoxins levels in the extrudates were determined using HPLC procedures, and the nutritional quality was assessed using in-vitro methods. The highest aflatoxin reduction in naturally contaminated peanut meal was 59% at feed moisture content of 35 g/100 g. Higher (91%) reduction was achieved in the artificially contaminated peanut meal at moisture of 20 g/100 g. In-vitro protein digestibility and Fluorodinitrobenzene (FDNB)-available lysine of the extrudates were not significantly different from non-extruded peanut meal. Extrusion conditions for aflatoxin reduction did not adversely affect protein nutritional quality.