The same extracts analyzed for total phenolic content and antioxidant activity by spectrophotometer was used for identification ofthephenoliccompounds by LC-ESI- qTOF-MS. Samples were filtered through a 0.22 mM nylon membrane filter (Merck Millipore Corporation, Germany). After the samples were prepared, 10 µ L was injected in a liquid chromatograph (UFLC, Shimadzu, Japan) coupled to a high-resolution mass spectrometer ofthe quadrupole type–flight time (Maxis Impact, Bruker Daltonics, Germany). A pre-column C18 (2.0 × 4 mm) and Luna C18 column (2.0 × 150 mm, 100 Å, 3 µ m) (Phenomenex Torrance, USA) were used for the chromatographic separation usingthe mobile phases: water acidified with 0.1% formic acid (eluent A) and acetonitrile acidified with 0.1% formic acid (eluent B). For separation, a gradient was used: 0–2 min, 10% B; 2–15 min, 10–75% B; 15–18 min, 90% B; 18–21 min 90% B; 21–23 min, 10% B, 23–30 min, 10% B, 0.2 mL.min -1 flow and the column temperature was set at 40 °C. The mass spectrometer was operated in the ESI negative modes with spectra acquired over a mass range of m/z 50 to 1200, with capillary voltage at 3.5 kV, nebulization gas pressure (N 2 ) of 2 bar, drying gas at 8 L min ”1 , source temperature 180 °C, RF collision of 150 Vpp; transfer 70 mS and pre- pulse storage of 5 mS. The equipment was calibrated with 10 mmol.L -1 sodium formate, covering the acquisition ran- ge of m/z 50 to 1200. Automatic MS/MS experiments were performed by adjusting the collision energy values as follows: m/z 100, 15 eV; m/z 500, 35 eV; m/z 1000, 50 e V, using nitrogen as the collision gas (Hoffmann et al., 2016). Data from MS and MS/MS were processed using Data analysis software 4.0 (Bruker Daltonics, Germany). Phenoliccompounds were characterized by the UV/Vis spectrum (210–800 nm), and the exact mass and MSn fragmentation patterns were compared to the equipment library data and databases (Metlin, MassBank, Kegg Compound, ChemSpider) and compared with the isotopic standard. The quantification ofphenoliccompounds were performed by external calibration curve with eight concentrations (0.039; 0.078; 0.156; 0.312; 0.625; 1.250; 2.50 and 5 µg.mL -1 ) with standards of each compound
In a Quality context, an experiment is a test that measures changes in system’s results, caused by overseen alterations in process’s input. Input variables, known as factors, are independent and may assume different values, the levels, these data may be of a quantitative or qualitative kind. Quantitative levels always need to be scalable (i.e. temperatures), while in the second case levels represent the variation of a condition, whether is to use or not an equipment, or trying out different materials in a process, etc. As a result ofthe procedure one or multiple responses, depending on the type of system, are obtained from each experiment. System’s performance is measured through comparison of changes in response values, as a reaction to factors’ manipulation. Hence, responses must be quantitative, in order to have well accurate and measurable indicators ofoptimization (Pereira & Requejo 2012).
Lipidic carbon sources (as vegetable oils) are cited as essential inducers for obtaining significant biosurfactant and lipase amounts (Dutra et al., 2008; Saharan et al., 2011). However, the mechanism that regulates biosynthesis widely varies for different microorganisms (Dalmau et al., 2000). In this study the lipase production was independent ofthe addition of lipids to the culture medium, achieving its maximum value of 3.89 U (Table 1) in the assay with the high level (+1) of ME and low levels (-1) of AG and SO. Colla et al. (2010) in their study obtained a maximum lipolytic activity of 4.52 U using soybean oil as a supplementary carbon source for Aspergillus spp. Sarkar and Laha (2013) obtained higher lipase production values when using glucose and olive oil as substrates and reduced lipolytic activity values were observed when using glucose as the sole carbon source. Higher soybean oil concentrations impacted negatively lipase production by Aspergillus niger and Aspergillus flavus according to Colla et al. (2016).
Bacteriocins from lactic acid bacteria are ribosomal synthesized antibacterial proteins/ peptides hav- ing wide range of applications. Lactobacillus pentosus SJ65, isolated from fermented Uttapam batter (used to prepare south Indian pan cake), produces bacteriocin having a broad spectrum of activity against pathogens. Optimization studies are of utmost important to understand the source of utiliza- tion and the conditions to enhance the production of metabolites. In the present study, an attempt was made to identify the parameters involved for maximal production of antimicrobial compounds espe- cially bacteriocin fromthe isolate L. pentosus SJ65. Initially, optimal conditions, such as incubation period, pH, and temperature were evaluated. Initial screening was done usingmethodology one- variable-at-a-time (OVAT) for various carbon and nitrogen sources. Further evaluation was carried out statistically using Plackett-Burman design and the variables were analyzed usingresponse sur- face methodologyusing central composite design. The optimum media using tryptone or soy peptone, yeast extract, glucose, triammonium citrate, MnSO 4 , dipotassium hydrogen phosphate and
Samples were prepared by weighing 20 g of grated Coalho cheese in 60 mL vials sealed with a polytetrafluoroethylene silicone septum (Supelco, Bellenfont, PA, USA). The system was submitted to an extraction temperature (T) of 20, 30, 45, 60, and 70 °C in a water bath and an equilibrium time (Eq) of 3, 10, 20, 30, and 37 minutes before fibre exposure. Prior to extractions, the fibre was conditioned to the gas chromatograph injector at the temperature of 270 °C for 60 minutes. Afterwards, the SPME fibre was exposed to the headspace, above the Coalho cheese sample for an extraction time (Ex), of 6, 20, 40, 60, and 74 minutes. Then the fibre was retracted into the needle and transferred to the gas chromatograph injector where analytes were desorbed. The values tested were all obtained from previous studies, in which they had yielded satisfactory results for SPME extraction in cheeses (Delgado et al., 2010; Hayaloglu et al., 2013; Kondyli et al., 2013).
Abstract The main objective ofthe present work was to investigate the potential of aqueous solutions of ionic liquids (ILs) and deep eutectic solvents (DES) as alternative solvents to extract value-added compoundsfrom biomass and related waste. These compounds include triterpenic acids (TTAs; ursolic, oleanolic and betulinic acids), a sesquiterpene lactone (cynaropicrin) and a phenolic compound (syringic acid), whereas the biomass samples investigated correspond to olive tree leaves, apple and pear peels, and cardo leaves. The interest on the described natural compounds is related to their wide variety of biological properties, with relevant applications in the food, cosmetic and pharmaceutical industries. The developed work was focused on developing more sustainable and cost-effective extraction/recovery strategies than those commonly used. To this end, a better understanding on the solubilisation mechanisms (by hydrotropic or micelle-based effects) was also searched. It was demonstrated the enhanced capacity of aqueous solutions comprising surface-active ILs to extract TTAs, achieving yields up to 2.5 wt.% fromolive tree leaves and up to 2.6 wt.% from apple peels, which are higher to those obtained with conventional organic solvents under similar conditions. An increase of 8 orders of magnitude in the solubility of ursolic acid in aqueous solutions of IL was verified when compared to pure water. Aqueous solutions ofsurface-active ILs were also demonstrated to be promising solvents to extract cynaropicrin from cardo leaves, leading to extraction yields up to 3.7 wt.% under the best identified conditions. Water was added as an anti-solvent, leading to the precipitation and recovery of 65 wt.% of cynaropicrin. Aqueous solutions of hydrotrope-based ILs were applied to extract of syringic acid from Rocha pear peels, leading to extraction yields up to 2.1 wt.% by reusing the solvent and up to 2.2 wt.% by reusing the biomass. These improvements in theextraction yield allowed to propose an extraction continuous process operating in countercurrent, in which the solvent and biomass are reused in a continuous mode. Taking advantage ofthe syringic acid solubility dependence with the IL concentration in aqueous solutions, where an enhancement up to 84-fold was obtained, water was again added as an anti-solvent allowing to recover 77 wt.% of syringic acid.
Different solid-liquid systems (maceration, microwave, ultra- sound, among many others) are available for theextractionof com- pounds. There is not a universal approach better than the others, focussing in reducing the time ofextraction, amount of solvents, the energy costs and the degradation patterns (Alonso-Salces et al., 2001; Dai & Mumper, 2010; Ince, Sßahin, & Sßümnü, 2013). Betacyanins are generally extracted by maceration extraction tech- nique with water as the main solvent, but aqueous organic solvent mixtures have shown certain improvements in the final extractions yields obtained. Maceration extraction is a conventional method easily transferable to industrial scale and traditionally used in theextractionof bioactive compounds. The main advantage is its sim- plicity, but if the variables are not properly optimized, very often requires long time periods and high temperatures resulting in high-energy costs and bioactive compounds degradation.
The obtained model coeﬃcients (Table 3) are empirical and cannot be associated with physical or chemical signiﬁcance. However, they are useful for predicting the results of untested extraction conditions . The sign ofthe eﬀect marks the performance oftheresponse. In this way, when a factor has a positive eﬀect, theresponse is higher at the high level, and when a factor has a negative eﬀect, theresponse is lower at the high level. The higher the absolute value of a coeﬃcient, the more important the weight ofthe corresponding variable. Based on the mathematical expressions (Table 2), no associations were found be- tween theresponse variables ofphenolic acids, ﬂavonoids, quercetin glycoside derivatives (Qgd), isorhamnetin glycoside derivatives (Igd) and kaempferol glycoside derivatives (Kgd). However, certain features regarding the general eﬀects ofthe variables are displayed. The re- levance ofthe signiﬁcant parametric values can be order as a function ofthe variables involved in a decreasing form as S > P ≫ t. Alexandre et al.  also found S as the most relevant variable on the HHP ex- traction of bioactive compoundsfrom pomegranate (Punica granatum L.) peels. Regarding the linear, quadratic, and interactive parametric eﬀects ofthe developed equations, it was found that they play an im- portant and signiﬁcant role in all evaluated responses. For the linear eﬀect, the variables P and S had strong values; meanwhile, the eﬀect of t was negligible in almost all cases. All independent variables had moderate quadratic or nonlinear eﬀects. Regarding the interactive ef- fects, the interactions ofthe variable t with the other variables (tP and tS) were of minor relevance; meanwhile, the PS interaction had a strong signiﬁcance in describing the behavior of almost all responses (with the exception of compound 10). The interactive parametric values of PS were accentuated in the responses of ﬂavonoids, Qgd, Igd, phenolic acids, and total phenoliccompounds. To make the combined eﬀects more explicit and to visually describe theextraction trends, the results were presented in theresponsesurface plots discussed below.
Based on the SEM images, sonication definitely had an important role in the explosive disruption ofthe physical structure of vegetal cell walls, and enhanced theextraction yield by the acoustic cavitation (37). Similar results have reported the effect of ultrasonic vibration on the physical structure of Achillea biebersteinii Afan. (21) and Euonymus alatus (Thunb.) Sieb. (38). The hydration process followed by the swelling process, also mass transfer ofthe soluble compoundsfromthe plant matrices to the solvent by osmotic and diffusion processes are two stages ofthe classical extraction process (21). In contrast, the UAE method enhances the swelling and softening process of cell walls via the hydration of pectinous material fromthe middle lamella. Consequently, it may lead to the breakup ofthe vegetal tissue during sonication (39) which means, UAE is much faster and more efficient than the classical extraction method. SEM images revealed that the efficiency of UAE for theextractionofphenoliccompounds, and consequently the enhancement of their antioxidant and antimicrobial activities could reasonably be considered superior to that ofthe classical extraction method.
Lizard tail (Houttuynia cordata Thunb.) is an Asian herb which has many biological activities, including antioxidative property from polyphenolic compounds. Responsesurfacemethodology and Box-Behnken design were employed to study the effect ofextraction temperature (30 to 70°C), extraction time (10 to 30 min), ethanol concentration (30 to 70%), and solvent to sample ratio (2 to 6 ml/g) on ultrasonic-assisted extractionofphenoliccompoundsfrom lizard tail and antioxidant capacity ofthe herb extract. Extraction temperature was the most relevant factor on the responses. Optimal condition was the extrac- tion temperature of 70°C for 30 min, using 60% ethanol concentration at the solvent to sample ratio of 5 ml/g. Model adequacies were confirmed by extraction at the optimal condition and normality of standardized residuals.
The preliminary range oftheextraction variables were determined through single factor experiments. Responsesurfacemethodology (RSM) based on central composite rotatable design (CCRD) was applied to evaluate the effects of four independent variables, enzyme amount (X 1 ), liquid/solid ratio (X 2 ), hydrolysis time (X 3 ), hydrolysis temperature (X 4 ), and their interaction on the measured response, extraction yield (Y). The independent variables were coded at five levels (-2, -1, 0, +1, +2), and the complete design consisted of 31 experimental points including 7 replications ofthe centre points. The coded levels ofthe independent variables used in the RSM design were listed in Table 1.
Perilla (Perilla frutescens), an aromatic vegetable, has been cultivated worldwide and used extensively for cooking and medicinal purposes in several Asian countries (Igarashi & Miyazaki, 2013; Li et al., 2015). The leaves of P. frutescens have been shown to have detoxicant, antitussive, antipyretic and antibiotic properties (Liu et al., 2013; Nakamura et al., 1998). Several studies have reported that extracts of perilla leaves have high antioxidant capacity, mostly due to the presence of polyphenolic compounds (Lee et al., 2013; Zhou et al., 2014). Phenoliccompounds are well-known for their beneficial health properties, attributed to their antioxidant and antiradical activities (Tao et al., 2014; Krishnaswamy et al., 2013; Heim et al., 2002). Moreover, rosmarinic acid (RA) has been shown to be the main biologically active polyphenolic compound in perilla leaves (Liu et al., 2013). RA is a well-known hydroxycinnamic acid ester that has interesting biological properties beneficial to human health, including antioxidant, anti-inflammatory, anticancer, and anti-allergenic activities (Lamien-Meda et al., 2010; Tang et al., 2014), and preventing food spoilage (Petersen & Simmonds, 2003; Pérez-Tortosa et al., 2012). Recently, interest has increased considerably in naturally occurring antioxidants for use in foods or medicinal materials as replacements for synthetic antioxidants such as BHA (beta hydroxy acid) and BHT (butylated hydroxy toluene), whose use is restricted due to safety concerns (Ito et al.,
Recently, reports on the application of ultrasound as a method for extractionof biolo- gical active compounds form plant material have been published. It has been showed that ultrasound-assisted extraction (UAE) technique can be especially suitable due to low equipment requirements and its high economic efficiency (12), offering at the same time high reproducibility, simplified manipulation, and reduced solvent and energy consump- tion (13). Beside these advantages, the usage of ultrasound for extraction diminishes the danger of thermal degradation of desired compounds and reduces significantly the time needed for the process itself (14). Cavitation, which occurs in the solvent due to the crea- tion, growth and implosion of gas bubbles (15) and mechanical effect of ultrasound which provides a greater penetration of solvent into cellular material (12, 14), are the main benefits ofusing this method. Like many other processes, this process also posses- ses certain disadvantages. They are manifested in the form ofthe effect causing the chan- ges in chemical composition and degradation of desired compounds, as well as in forma- tion of free radical species inside of gas bubbles (16).
OPTIMIZATIONOF SAPONIN EXTRACTION CONDITIONS FROM Ampelozizyphus amazonicus BY USING EXPERIMENTAL DESIGN AND SURFACERESPONSEMETHODOLOGY. This works describes the use of experimental design and surfaceresponsemethodology for optimizationof saponin extractionfrom Ampelozizyphus amazonicus. For this purpose, a method employing extraction based on maceration assisted by ultrasound technique was utilized. The following factors were studied: extraction length of time and solvent composition. The total saponin was determined by using a gravimetric method and the results expressed by their relative proportion to total crude extract. For the specific condition, 60% hydro-alcoholic solution and 18 minutes extraction length of time has shown the best results. This method can be useful for extractionof substances with biological importance.
different aeration time (2, 10 and 18 h). As can be seen in Figures 3a-3c, almost the same trends were found as the aeration time changed from 2 to 18 h. It is clear fromthe figures and Eq. (2) that the most significant factor on theresponse is aeration time. So that, as the aeration time increased theresponse was increased. It should be noted that the aeration time did not have significant effect on theresponse at the values more than 10 h. It is proven by perturbation plot (Figure 4a). The perturbation plot shows the com- parative effects ofthe variables on theresponse. A steep curvature in aeration time, C curve, shows that theresponse was very sensitive to this factor. The comparatively semi-flat A and B curves show less sensitivity ofthe COD removal efficiency to alter with respect to a change in aeration time. In other words, the COD in and biomass concentration (in the range
Lipase assay was done spectrophotometrically using p-nitrophenyl palmitate (procured from Sigma) as the substrate. The assay mixture contained 1ml of 16.5 mM solution of p- nitrophenyl palmitate in 2-propanol along with Tris-HCl buffer, pH 8 (supplemented with 0.1% arabic gum and 0.4% Triton X-100) in a ratio of 1:9. The enzyme solution (0.025 ml) was added to it and incubated in water bath at 37°C for 10 min. p-nitrophenol was liberated from p-nitrophenyl palmitate by lipase mediated hydrolysis imparting a yellow color to the reaction mixture. After incubation, 2 ml of dist. water was added and the absorbance was measured at 410 nm (Kordel et al., 1991). Absorbance of control was also recorded. One unit (U) of lipase activity was defined as the amount of enzyme that liberates one micromole of p-nitrophenol, per min under the assay conditions.
All fermentations were performed in a stirred tank bioreactor using a standard medium supplemented with a fixed concentration of carbon source as described previously (Stergiou et al. 2012). The experimental scales ofthe operating variables (pH and temperature) are depicted in Tables 1 and 2. Cell growth and α -amylase activity assays were carried out at regular intervals in aliquots of 5.0 mL which were withdrawn fromthe culture medium (Stergiou et al. 2012).
The biochemical and nutritional cell environment in a bioreactor is strongly influenced by growth conditions and medium composition, namely carbon and nitrogen sources, mineral salts, trace elements, peptides, amino acids, vitamins and/or other factors. Therefore, theoptimizationof culture medium composition is essential for the effective production in bioreactor [ 4 , 5 ]. The production of biomolecules, such as enzymes for food industry, often requires the use of complex and cheap industrial media based on yeast extract (YE) and bactopeptone as nitrogen sources. Complex organic nitrogen sources are less expensive than pure amino acids and nitrogen bases and, additionally, can supply a variety of vitamins, minerals and other growth co-factors. Furthermore, cell growth is generally more rapid and efficient when these nutrients are used, since they reduce the number of components that cells would otherwise have to synthesize de novo. A widely used complex rich growth medium containing YE and bactopeptone as nitrogen sources and glucose as carbon source played an important role in the efficiency and economics ofthe fermentation process [ 6 ]. The activity and stability of enzymes is influenced by the type of strain, cultivation conditions (temperature, pH, aeration, agitation, incubation time) and culture medium composition (particularly carbon and nitrogen sources) [ 7 – 9 ].
TheResponseSurfaceMethodology (RSM) is important in designing and analyzing products and processes. It is optimization study for existing studies and products. The most common applications of RSM are in Industrial, Biological and Clinical Science, Social Science, Food Science, Physical and Engineering Sciences. Responsesurfacemethodology will give a solution in either 2-Dimensional or 3 Dimensional to get a perfect solution for the parameter setting to get optimal solution for a response .
methods which involve electron transfer reaction mechanism. This classification can explain the high Pearson’s correlation coefficients shown in Figure 1 because these methods act through the same mechanism. On the other hand, the ORAC method is based on hydrogen atom transfer, this may explain the low value ofthe correlation coefficient between TPC and ORAC. The same was reported by Gonçalves, Lajolo and Genovese  and Isabelle et al. . Anthocyanins are responsible for the red to purple to black pigments found in fruits and vegetables  and they are the largest group of water-soluble pigments in the plant kingdom that belong to the class ofphenoliccompounds [38,39]. Fromthe results shown in Figure 2, can be observed that there were good relationships be- tween TPC and ACC for black mulberry (R = 0.9345), blackberry (R = 0.8007) and strawberry (R = 0.7560).