Optimization of ultrasonic-assisted extraction process of Poria cocos polysaccharides by response surface methodology

Polysaccharides production from Poria cocos was carried out using aqueous NaOH with the assistance of ultrasonic. Experimental design was used to investigate the effect of three parameters (extraction time, extraction concentration of NaOH, and ratio of aqueous NaOH to raw material) on polysaccharides yields. The ranges of the factors investigated were 1–3 min for extraction time (X₁), 0.5–1.0 mol/L for extraction concentration of NaOH (X₂), and 30–50 for ratio of aqueous NaOH to raw material (X₃). The statistical analysis of the experiment indicated that extraction concentration of NaOH had significant effect on P. cocos polysaccharides yields. The central composite design showed that polynomial regression models were in good agreement with the experimental results with the coefficients of determination of 0.9935 for P. cocos polysaccharides yield. The optimal condition for P. cocos polysaccharides yield within the experimental range of the variables studied was at 2.44 min, 0.789 mol/L, and 53.0. At this condition, the predicted yield of polysaccharides extracted was 82.3%.     

Orthogonal test design for optimization of the extraction of polysaccharides from Phascolosoma esulenta and evaluation of its immunity activity

Yield of polysaccharides from Phascolosoma esulenta obtained by phosphate buffer extraction through an orthogonal experiment (L₉(3)⁴) were investigated to get the best extraction conditions. The results showed that extraction temperature, ratio of phosphate buffer to raw material, extraction time, and ratio of trypsinase to raw material were the main four variables that influenced the yields of extracts. The highest yield was obtained when extraction temperature, ratio of phosphate buffer to raw material, extraction time and ratio of trypsinase to raw material were 40 °C, 2, 5.5 h and 1.6, respectively. The immunity-stimulating method showed that polysaccharides from P. esulenta could significantly raise liver, spleen and thymus index of mice and enhance Con A-stimulated mouse spleen cells proliferation. These results indicate that polysaccharides from P. esulenta had significantly higher immunity-stimulating activities.     

Optimization of extraction technology of the Anemone raddeana polysaccharides (ARP) by orthogonal test design and evaluation of its anti-tumor activity

In order to get high quality bioactive polysaccharides (ARP) from Anemone raddeana, an orthogonal experiment (L₉(3)⁴) was applied to optimize the best extraction conditions. Extraction time, extraction temperature, number of extraction and water to raw material ratio were the main factors to influence the yield and purity of the extracted crude polysaccharides. The four factors chosen for the present investigation were based on the results of a single-factor test. The optimum extraction conditions were determined as follows: extraction time 1.5 h, temperature 80 °C, number 4 and water to raw material at 6. Under optimized conditions, the experimental yield 7.97 ± 0.27% agreed closely with the predicted yield. In vivo ARP at suitable dose is effective on Th1 cells, as associated with an enhancement of IgG2a and IgG2b levels. In vitro ARP significantly inhibited the proliferation of human hepatocellular carcinoma HEP-G2 and human intestinal cancer HCT-22 cells, indicating ARP could be a potential anti-cancer therapeutic agent.     

Mixotrophic growth ofChlorella sorokiniana in outdoor enclosed photobioreactor

Chlorella sorokiniana was cultured in heterotrophic or mixotrophic mode in outdoor enclosed tubular photobioreactor. The culture temperature was maintained at 32–35 °C. At night, theChlorella culture grew heterotrophically, and 0.1 M glucose was completely consumed. The biomass growth yield of glucose was 0.35 ± 0.001 g-biomass g-glucose^–1. During the day, the algal culture grew mixotrophically and the biomass growth yield was 0.49 g-biomass g-glucose^–1 in low density culture (initial biomass concentration, X_o = 2 g l^–1), 0.56 g-biomass g-glucose^–1 in medium density culture (X_o = 4 g l^–1) and 0.46 g-biomass g-glucose^–1 in high density culture (X_o = 7 g l^–1). The daily area productivity of the culture, with X_o = 4 g l^–1 corresponded to 127 g-biomass m^–2 d^–1 during the day and 79 g-biomass m^–2 d^–1 during the night. In all the cultures, the dissolved O₂ concentration increased in the morning, reached the maximum value at noon, and then decreased in the afternoon. The dissolved CO₂ concentration remained at 3 mBar in the morning and increased in the afternoon. Glycolate was not found to accumulate in culture medium.     

Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 g g^–1 fresh weight) with high quality (A_260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies.     

Dodonaea viscosa var. angustifolia leaf: New source of polysaccharide and its anti-oxidant activity

Ultrasonic technology was applied for polysaccharide extraction from the leaves of Dodonaea viscosa and response surface methodology (RSM) was used to optimize the effects of processing parameters on polysaccharide extraction yield. Three independent variables were extraction time (X₁), extraction temperature (X₂) and ultrasonic power (X₃), respectively. The statistical analysis indicated the independent variables (X₁, X₂, X₃), the quadratic terms (X₁₁ and X₃₃) and the interaction terms (X₁X₂, X₁X₃, X₂X₃) had significant effects on the yield of polysaccharides (P < 0.05). The optimal extraction conditions of D. viscosa leaf were determined as follows: extraction time 50.54 min, extraction temperature 85 °C and ultrasonic power 400 W. Under these conditions, the experimental yield of polysaccharides was 9.455 ± 0.24%, which was agreed closely with the predicted value (9.398%). The evaluation of anti-oxidant activity suggested that the polysaccharide exhibited significant protection against DPPH and hydroxyl radicals and could be explored as a nutraceutical agent.     

Culture of Hypnea musciformis (Rhodophyta,Gigartinales) on artificial substrates attached to linear ropes

Hypnea musciformis is the only species so far exploited in Brazil as raw material for the production of k-carrageenan. Due to the erratic production in space and time, increasing harvest and transportation costs, experiments have been performed in order to assess the viability of H. musciformis mariculture.In nature the species occurs as an epiphyte, and so mariculture using artificial substrates that simulated the natural host of the species was tried. These substrates were attached, at regular intervals, to linear ropes. In the sea, these ropes were stretched between cement blocks.Seeding occurs naturally, by means of spores, or detached pieces of H. musciformis scattered in the water column that get entangled on the ropes. The best yields (0.54 wet kg m^–1 month^–1) were obtained with unthreaded rope substrates maintained in a vertical position by small rafts. Production is highest in the first 18 m off the rocky shore (0–2.1 m deep), at the highest substrate density utilized (10 m^–1), 2–3 months after installing the ropes in seawater. The main factor controlling seasonal production is water movement.     

Optimization of polysaccharides (ABP) extraction from the fruiting bodies of Agaricus blazei Murill using response surface methodology (RSM)

Response surface methodoloty (RSM) was used to optimize the extraction conditions of polysaccharides (ABP) from the fruiting body of Agaricus blazei. A central composite design (CCD) was used for experimental design and analysis of the results to obtain the optimal processing parameters. Four independent variables such as extraction temperature (°C), ratio of water to raw material, number of extraction, and extraction time (h) were investigated. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical methods. The 3-D response surface plot and the contour plot derived from the mathematical models were applied to determine the optimal conditions. The optimum extraction conditions were as follows: extraction temperature 91 °C, ratio of water to raw material 14, number of extraction 6, and extraction time 2.1 h. Under these conditions, the experimental value was 65.8 ± 1.42, which is well in close agreement with value predicted by the model.     

Culture ofGelidium sesquipedale (Clem.) Born.et Thur. in a chemostat system. Biomass production and metabolic responses affected by N flow

Gelidium sesquipedale is the most important raw material used for extraction of agar in Spain. Based on chemostats, a system of culture for macroalgae with a continuous flow of culture medium has been developed. A stressed morphotype from the South of Spain was cultured, and the effects of different rates of NO ₃ ^– flow on growth and internal constituents were investigated in the laboratory. Cultivation was successful after optimizing factors affecting growth, such as irradiance level, renewal rate and water movement. Mass production was dependent on N supply. With a flow of 35 mol NO₃ ^– g^–1 DW d^–1, optimal values of growth (2.1% d^–1) and biomass yield were obtained. In these conditions, biomass yield resembled the values observed in natural populations (about 500 g DW m^–2 y^–1). When the flow of N was reduced to 15 mol NO ₃ ^– g^–1 DW d^–1, growth rate and biomass yield were reduced three-fold, and were null when N was supplied as 7 mol NO ₃ ^– g^–1 DW d^–1. C:N ratio was an index of the physiological status of the tissue, remaining low when N was sufficient and raised to critical values when N supply was limited. Phycobiliproteins, kept at a constant irradiance level, were affected by N supply, acting as an internal nitrogen reserve, unlike chlorophylla. An effective phycobiliprotein synthesis took place when the flow of N was sufficient. Agar yield, on dry weight basis, was similar as a function of N flow, whereas agar yield of the culture was higher when N was sufficient as a result of growth not being limited by N.This system of culture, commonly used in microalgal studies, may have an important use in macroalgae as a system to obtain biomass of high quality as well as a good tool for physiological studies in conditions of continuous and controlled flow of nutrients.     

Sequential liquid-liquid extraction of d-amino acid oxidase from Trigonopsis variabilis

A method for isolation of d-amino acid oxidase (DAAO) from disrupted Trigonopsis variabilis cells has been developed. In an aqueous two-phase system consisting of PEG6000 (220 g l^–1), potassium phosphate (110 g l^–1, K₂HPO₄ + KH₂PO₄ = 10.1:1, mol mol^–1) and dl-methionine (11 g l^–1), the major portion of cellular proteins (87%) was partitioned into the salt phase. By sequential extraction, 48% of DAAO was recovered in PEG phase, giving a yield of 211 U mg protein^–1.     

A simple method for DNA extraction from sporophyte in the brown alga <Emphasis Type="Italic">Laminaria japonica</Emphasis>

A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA was purified using a chloroform-isoamyl alcohol (24:1 v/v), and precipitated in cold isopropanol. The yield was from 18.7 to 37.5 μg g⁻¹ (wet weight) and the purity of total DNA was determined spectrophotometrically as the ratio of A₂₆₀/A₂₈₀, which was about 1.7–1.9. The extracted DNA was of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion. This method is a reproducible, simple, and rapid technique for routine DNA extraction from sporophyte in Laminaria japonica. Furthermore, the low cost of this method makes it attractive for large-scale studies.     

Response surface optimization of polysaccharides extraction from liriope roots and its modulatory effect on sjogren syndrome

The response surface methodology (RSM) was employed to study the extraction of liriope polysaccharides. The maximum extraction rate (64.1%) of liriope polysaccharides obtained by using the above optimised concentrations of the variables was extraction time (X₁) at 185 min, ratio of liquid to solid (X₂) at 11, and extraction temperature (X₃) at 94 °C. Forty male Wistar rats were used in the present experiment. After 2 weeks of acclimatization, animals were divided into 5 equal groups: control, SS model and 3 polysaccharides-treatment groups. Rats were orally administered their respective doses every day for 6 weeks. Liriope polysaccharides significantly increased the amount of salivary secretion, and the relative weight of spleen, thymus and submandibular glands. Therefore, the present results revealed that liriope polysaccharides exert a protective effect against tissue damage in rats with sjogren syndrome (SS).     

Extraction of crude polysaccharides from Duchesnea indica (Andrews) Focke: optimization by response surface methodology

A full set of optimization procedure was applied to the extraction of anti-viral polysaccharides from Duchesnea indica (Andrews) Focke. By Plackett–Burman factorial design, three parameters (extraction time, extraction temperature, and ratio of water to raw material) were identified as significant to the extraction yield. However, no significant parameters had been identified for antiviral activity. A three-level-three-factor Box–Behnken factorial design was then employed to further optimize the extraction condition. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis and also examined using appropriate statistical methods. This led to the construction of a response surface indicating the optimal values for each parameter and response studied. Concerning the extraction yield, an extraction at 98.51?ºC for 6.16 h with a ratio of water to raw material of 30.94 mL/g was found to be optimal. Under the optimized conditions, the experimental yield was 6.430 ± 0.078%, which was well matched with the predicted yield of 6.509%.     

Micropropagation of licorice (Glycyrrhiza glabra L.) through shoot tip and nodal cultures

Summary Apical and axillary buds ofGlycyrrhiza glabra commonly known as licorice, a plant of repute in the Indian system of medicine, were used for induction of adventitious shoots. For induction of multiple shoots, Murashige and Skoog’s (MS) medium with N⁶-benzyladenine (BA, 0.88–8.87 μM) was used. Reduction in major salts of MS medium enhanced the multiplication ratio up to 1∶10. Plants transferred to the greenhouse showed 90% survival. The present work describes a stepwise protocol for production ofGlycyrrhiza glabra plants on simple minimal media, where very high multiplication rates with healthy root systems were obtained. Roots being the organ of commercial importance, the protocol has tremendous potential.     

Modelling continuous culture of Rhodospirillum rubrum in photobioreactor under light limited conditions

Rhodospirillum rubrum was grown continuously and photoheterotrophically under light limitation using a cylindrical photobioreactor in which the steady state biomass concentration was varied between 0.4 to 4 kg m^–3 at a constant radiant incident flux of 100 W m^–2. Kinetic and stoichiometric models for the growth are proposed. The biomass productivities, acetate consumption rate and the CO₂ production rate can be quantitatively predicted to a high level of accuracy by the proposed model calculations. Nomenclature: C _X, biomass concentration (kg m^–3) D, dilution rate (h^–1) Ea, mean mass absorption coefficient (m² kg^–1) I , total available radiant light energy (W m^–2) K, half saturation constant for light (W m^–2) R _W, boundary radius defining the working illuminated volume (m) r _X, local biomass volumetric rate (kg m^–3 h^–1) <r _X>, mean volumetric growth rate (kg m^–3 h^–1) V _W, illuminated working volume in the PBR (m^–3). Greek letters: , working illuminated fraction (–) _M, maximum quantum yield (–) bar, mean energetic yield (kg J^–1).     

In situ monitoring of chlorophyll fluorescence to assess the synergistic effect of low temperature and high irradiance stresses inSpirulina cultures grown outdoors in photobioreactors

A chlorophyll fluorescence technique was applied to anin situ study on the effects of low temperature and high light stresses onSpirulina cultures grown outdoors in controlled tubular photobioreactors at high (1.1 g L^–1) and low (0.44 g L^–1) biomass concentrations. Diurnal changes in PSII photochemistry (F _v/F _m) after 15 min of darkness, or in the light (dF/F _m), and non-photochemical (qN) quenching were measured using a portable, pulse-amplitude-modulated fluorometer. The depression of theF _v/F _m ratio ofSpirulina cultures grown outdoors at 25°C (i.e. 10°C below optimum for growth) and 0.44 g L^–1, reached 30% at the middle of the day. At the same time of the day thedF/F _m ratio showed a reduction of up to 52%. The depression of bothF _v/F _m anddF/F _m was lower in the cultures grown at 1.1 g L^–1. Photoinhibition reduced the daily productivity of the culture grown at 0.44 g L^–1 and 25°C by 33% with respect to that grown at 35°C. Changes in the growth yields of the cultures grown under different temperatures and growth rates correlate well with analogous changes in photon yield (dF/F _m). Simple measurements of photochemical yield (F _v/F _m) can be used to test the physiological status ofSpirulina cultures. The results indicate that the saturating pulse fluorescence technique, when usedin situ, is a powerful tool for assessment of the photosynthetic characteristics of outdoor cultures ofSpirulina.     

Influence of medium composition and aeration on the synthesis of biosurfactants produced by Candida antarctica

Candida antarctica synthesised surface-active mannosylerythritol lipids at 46 g l^–1 by adding 80 g soybean oil l^–1 to the medium and maintaining the pO₂ at 50% with an air flow rate 1 vvm. Two-stage culturing of C. antarctica avoided medium foaming but the yield of biosurfactants synthesis was 28 g l^–1. The biosurfactants decreased the surface tension of water to 35 mN m^–1.     

Influence of operational parameters in the flocculation and production ofLactobacillus plantarum

Summary The influence of different operational parameters, such as the dilution rate (D) and the bleeding rate (B), in the production of a flocculent strain ofLactobacillus plantarum was studied. The effect of the dilution rate was demonstrated to be related to the lactic acid concentration inside the reactor. The effect of the bleeding rate was shown to be critical in the stabilization of the operation (due to a better pH control). It also allowed a continuous recovery of cells outside the reactor. Viability testing of the lactic starter cultures showed that operation with cell purge increased the viability of the starter cultures obtained.Nomenclature B Bleeding rate, h^–1 - D Dilution rate, h^–1 - F Feed flow rate, L h^–1 - I Feed velocity, m h^–1 - Specific growth rate, h^–1 - v Lactic acid specific productivity, g g^–1 h^–1 - P Product concentration (lactic acid), g L^–1 - P _out Product concentration leaving the system, g L^–1 - Q _b Bleeding flow rate, L h^–1 - R Recirculation velocity, m h^–1 - S Substract concentration, g L^–1 - t Time, h - T _p Time of ascensional flow (length of the column/total ascensional velocity), h - T _r Residence time (1/D), h - V Volume of the reactor, L - X Cell concentration, g L^–1 - X _out Cell concentration leaving the system, g L^–1     

A model for growth of Artemia franciscana cultures based on food ration-dependent gross growth efficiencies

Laboratory cultures of Artemia franciscana grown under batch regimes at constant temperatures (28 °C) and salinity (35 g l^–1), three initial food concentrations (0.1, 0.4 and 1 M cells ml^–1), various daily food rations (0.1–9M Dunaliella tertiolecta cells Artemia ^–1), and different population densities (1–16 ind ml^–1) were used to develop a model of population growth. Growth rates and gross growth efficiencies (K ₁) were largely independent of population densities and initial food concentrations but determined by age and daily amount of food ingested. While maximum growth rates were found with the highest rations, K _{1 max} peaked at rations of 0.5 million cells d^–1 and decreased at feeding levels above this. A contour plot showing the trend relating K ₁ to Artemia size and ingestion rate in combination and was used to model growth in analogous controlled feeding conditions. Computer simulations using this model paralleled published results of final 15-day average individual sizes of Artemia. Optimal results for near constant food utilization are predicted for high initial population densities (100 Artemia nauplii ml ^–1) and daily culls of enough animals to equilibrate food demand with food availability. This strategy could permit a range of Artemia sizes harvested, maximize final individual sizes and retain high total yields (> 1.2 kg dry wt 1^–1). Effects of different culture strategies are discussed.