Assessment of Carotenoids Recovery through Cell Rupture of <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> CBS 2636 Using Compressed Fluids

The increasing demand for carotenoids by industries has drawn attention to their bio-production. Since pigments are intracellular, extraction steps are then needed after cell cultivation. In this work, different strategies for extraction of carotenoid pigments from Sporidiobolus salmonicolor (CBS 2636) were investigated. The cell rupture was carried out using dimethyl sulfoxide (DMSO), two pure compressed fluids, supercritical carbon dioxide and propane, and also a combination of pressurized fluid treatment followed by liquid DMSO. Dichloromethane, ethanol, ethyl acetate, and acetone were tested for the carotenoids extraction. Results obtained show that when multiple solvents were used a synergetic effect on the extent of carotenoids recovery was verified. Maximum concentration of total carotenoids (2,875 μg/L) was obtained in the treatment using supercritical CO₂ (300 bar/120 min) followed by dimethyl sulfoxide to disrupt the cell, and then the extraction with a solution of acetone/methanol (7:3, v/v).     

Assessment of hydrolysis of cheese whey and use of hydrolysate for bioproduction of carotenoids by Sporidiobolus salmonicolor CBS 2636

BACKGROUND: The increasing industrial demand for carotenoids has led to growing interest in their bioproduction. The need to reduce production costs has encouraged the use of low‐cost agroindustrial substrates. In this context, this work studied the pretreatment of Mozzarella cheese whey and the use of the pretreated whey as fermentation medium for the bioproduction of carotenoids by Sporidiobolus salmonicolor CBS 2636. RESULTS: Bioproduction was carried out in an orbital shaker using a 10 mL L^?1 inoculum, incubation at 25 °C and a stirring rate of 180 rpm for 120 h in a non‐illuminated environment. The carotenoids were recovered using liquid N₂ combined with dimethyl sulfoxide for cell rupture and an acetone/methanol mixture (7:3 v/v) for extraction. The maximum concentration of total carotenoids obtained was 590.4 µg L^?1 in a medium with 900 g L^?1 cheese whey hydrolysate and 4 g L^?1 K₂HPO₄ at 180 rpm, 25 °C and pH 4. CONCLUSION: The use of enzyme‐hydrolysed cheese whey was more effective in carotenoid bioproduction by S. salmonicolor CBS 2636 than the use of acid‐hydrolysed cheese whey. The concentration of total carotenoids obtained with the enzymatic hydrolysate was six times higher than that obtained with the acid hydrolysate. Copyright © 2009 Society of Chemical Industry     

Functional food oil coloured by pigments extracted from microalgae with supercritical CO2

A functional food oil, rich in fatty acids and antioxidants, coloured with pigments (carotenoids) extracted with supercritical CO₂ from the microalga Chlorella vulgaris, was produced, having in view its use in food industry (namely for derived seafood). The supercritical fluid extraction (SFE) was carried out in order to study the effect of several modifiers (oil mixed with the microalga and ethanol with the supercritical CO₂), the degree of crushing of the microalga and the supercritical fluid flow rate, at a pressure of 300 bar and temperature of 40 °C. Moreover, the microalga pigments were also extracted with acetone and with vegetable oil at room and high temperature. The recovery of carotenoids was 100% with oil at room temperature for 17 h, 70% with oil at 100 °C for 30 min, 69% with supercritical CO₂ at 40 °C and 300 bar. In SFE the degree of crushing strongly influenced the extraction recovery and higher pigment recoveries were obtained with well crushed biomass.     

Evaluation and Improvement of Extraction Methods for the Analysis of Aflatoxins B1, B2, G1 and G2 from Naturally Contaminated Maize

The extraction procedure for aflatoxin determination in maize is based on a methanol–water (8 + 2 v/v) or an acetone–water (85 + 15 v/v) mixture. Initially, the extraction efficiency of two solvents was evaluated for each aflatoxin. Different results were obtained for highly contaminated maize: significantly higher levels of aflatoxin B₁ were obtained by acetone–water, on the contrary higher levels of aflatoxin G₂ were achieved by methanol–water. Then, acetone–water mixtures in different proportions (7 + 3, 6 + 4 and 5 + 5 v/v) were tested to improve the extraction of aflatoxin G₂. Applying these extraction mixtures, the values both of aflatoxin B₁ and of other aflatoxins were generally higher compared to those obtained by acetone–water 85 + 15; moreover, acetone–water (6 + 4) and (7 + 3) showed the best extraction efficiency for all aflatoxins.     

Effect of extraction parameters on the carotenoid recovery from tomato waste

Tomato waste is an important source of natural carotenoids. This study was carried out to assess the extractability of tomato waste carotenoids in different organic solvents and to optimise the extraction parameters (type of solvent, extraction time, temperature and extraction steps) for maximum yield. Among other solvents, we tested a new environmentally friendly one, ethyl lactate, which gave the highest carotenoid yield (243.00 mg kg^?1 dry tomato waste) at 70 °C, compared to acetone (51.90 mg kg^?1), ethyl acetate (46.21 mg kg^?1), hexane (34.45 mg kg^?1) and ethanol (17.57 mg kg^?1). The carotenoid recovery was significantly (P < 0.05) affected by the number of extraction steps and temperature in all solvents. Mathematic equations predicted rather satisfactorily (R² = 0.89–0.93) the rate of carotenoid extraction in the above‐mentioned solvents. Carotenoid concentration increased with time, approaching a quasi‐saturated condition at approximately 30 min of extraction.     

Thermal and light stabilities and antioxidant activity of carotenoids from tomatoes extracted using an ultrasound-assisted completely solvent-free method

The use of petroleum-derived solvents, particularly volatile organic compounds (VOCs), in the chemical industry has increased the contamination and residual effects of these solvents. Ionic liquids (ILs) can potentially replace VOCs, thereby reducing the risks of environmental contamination and toxicity. In this context, the objectives of this study were as follows: 1 — to obtain an ionic liquid for use in extracting carotenoids from tomatoes with ultrasound assistance; and 2 — to determine the stability and antioxidant activity of tomato carotenoid extracts. Ultrasound can also efficiently extract carotenoid compounds with ionic liquids in comparison with conventional VOC solvents (obtained from an all-trans-lycopene 7.5–8.0 μg·g^− 1 tomato sample by IL and 6.2–7.7 μg·g^− 1 by acetone). Similarly, the activation energies (E_a) in aqueous medium were obtained for the IL carotenoid extract (10.8 kJ·mol^− 1) and acetone carotenoid extract (9.4 kJ·mol^− 1). The antioxidant activities of the tomato carotenoid extract were 7.4 and 12.4 relative to α-tocopherol for the ionic liquid extract and acetone extract, respectively. The combination of chromatographic analysis and degradation kinetics provided data for positive assessment similarity of thermal and light stabilities of tomato carotenoids extracted by IL and extracted by acetone.     

Carotenoid reaction with free radicals in acetone and toluene at different oxygen partial pressures An ESR spin-trapping study of structure–activity relationships

 The influence of eight commonly occurring carotenoids on (1) the production of spin adducts, (2) the hyperfine splitting constants a _H and a _N of the spin adduct electron spin resonance (ESR) spectra, and (3) the production of the oxidized N-tert-butyl-α-phenylnitrone (PBN) derivative N-benzoyl-N-tert-butylnitroxide (PBNOx) was investigated in ESR spin-trapping experiments, in which radicals were thermally generated from 2,2^′-azobis(2,4-dimethylvalero nitrile) (AMVN) and trapped by PBN. In the polar solvent acetone under air, all carotenoids except β-carotene were shown to diminish the number of spin adducts formed by about 20%. A prooxidative effect of β-carotene was even demonstrated at a higher concentration. In the less polar solvent toluene under air, most carotenoids lowered the amount of spin adducts, but effects were not as large as in acetone, and differences were found between (1) the more effective carotenoids with two cyclohexene rings conjugated to the polyene backbone (and lycopene), and (2) carotenoids with zero or one conjugated cyclohexene ring. The formation of PBNOx in toluene, which was essentially due to breakdown of peroxyl spin adducts, was significantly inhibited by astaxanthin, canthaxanthin and lycopene. Throughout, only alkoxyl radical adducts of PBN were observed. However, a significant difference was found between the a _H values of zeaxanthin and the carbonyl carotenoid astaxanthin, indicating structural differences of the spin adducts. Received: 15 April 1996     

Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from the microalga Haematococcus pluvialis

Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from Haematococcus pluvialis was carried out, for several experimental conditions, using a semi-continuous apparatus. The microalga was previously freeze-dried and ground with a ball mill. The effects of pressure (200 and 300 bar), temperature (40 and 60 °C), degree of crushing, as well as the use of ethanol as a co-solvent (10%) on the extraction efficiency were assessed. Organic solvent extractions, using acetone, were also carried out in a vortex, on ground cells mixed with very small glass beads. Supercritical extraction from the completely crushed alga was compared with acetone and the highest recovery of carotenoids (92%) was obtained at the pressure of 300 bar and the temperature of 60 °C, using ethanol as a co-solvent.The extraction recovery increased with the pressure at 60 °C. On the other hand, the increase in temperature, at 300 bar, led to a slight improvement. The main carotenoid of Haematococcus pluvialis is the esterified astaxanthin (about 75%). Other carotenoids present are lutein, astaxanthin (free), β-carotene and canthaxanthin. All of them were recovered through supercritical fluid extraction with values higher than 90%, with the exception of canthaxanthin (about 85%), at a pressure of 300 bar and a temperature of 60 °C.     

Recovery of lycopene from industrially derived tomato processing by-products by pulsed electric fields-assisted extraction

The influence of pulsed electric fields (PEF) pre-treatment at different field strength (E = 1–5 kV/cm) and energy input (W_T = 5–10 kJ/kg) on the recovery yield of lycopene in either acetone or ethyl lactate from industrial tomato peels residues, was investigated. The rate of lycopene extraction in both solvents decreased with time and was predicted rather satisfactorily (R² = 0.96–0.99) by the Peleg's model. Micrograph of tomato peels showed that PEF induced size reduction and separation between the plant cells likely due to pore formation and leakage of intracellular matter. Coherently, PEF treatment (5 kV/cm, 5 kJ/kg) significantly enhanced the extraction rate (27–37%), the lycopene yields (12–18%) and the antioxidant power (18.0–18.2%) in either acetone and ethyl lactate extracts, as compared with untreated samples. However, acetone gave the highest lycopene yield. HPLC analyses revealed that all-trans lycopene was the main carotenoid extracted and no degradation/isomerization phenomena occurred. The results obtained in this work suggest that the application of PEF prior to solid-liquid extraction with environmentally friendly solvents could represent a sustainable approach for the valorization of industrial tomato peels residues.Industrial relevanceIndustrial processing of tomatoes generates large amount of by-products, mainly peels, which represent a cheap and abundant source of natural carotenoids, especially lycopene. The recovery lycopene from tomato peels residues is a crucial step for use in a wide range of industrial applications in food, cosmetic and pharmaceutical sectors as natural pigment and antioxidant. PEF pre-treatment allows to intensify the extractability of lycopene from of tomato processing by-products using environmentally friendly solvents, thus adding new value to the tomato processing chain, improving economic performances and decreasing waste problems.     

Effect of extraction method and ripening stage on banana peel pigments

Carotenoids are one of the most widespread pigments in nature and can be used as health‐promoting natural food colorants. Banana peel, which is a by‐product of banana processing, contains a range of bioactive compounds including carotenoids. There is no published research on the extraction of food‐grade carotenoids from banana peels. This study evaluated the change in the banana peel carotenoid content over its ripening stages and determined the best possible solvent to extract carotenoid for food applications. The solvents permitted under Food Standard Australia New Zealand were used in the study. Ripeness stage 5 contained the highest content of total carotene at 1.86 μg g^?1 of banana peel. From one gram of banana peel, 0.57 μg of xanthophyll and 0.84 μg of beta‐carotene were extracted from ripening stage 5 with a solvent combination of hexane–diethyl ether–acetone and hexane–diethyl ether, respectively.     

Application of carbon dioxide in subcritical state (LCO2) for extraction/fractionation of carotenoids from red paprika

Carbon dioxide, as a liquid (LCO₂) in subcritical state, was applied for extraction of carotenoids from ground paprika. The increasing polarity of LCO₂ with the decrease of its temperature enabled fractionation of pigments according to their increasing polarity. The main constituents of the fractions of +6 °C and −6 °C were triacylglycerols (TAGs) including a small concentration of β-carotene. At −16 °C, more polar pigments (capsorubin, capsanthin, zeaxanthin) and their fatty acid (FA) esters were extracted. The pre-concentration ratio of carotenoids in the fraction at -16 °C was 17.2 with respect to Fraction at +6 °C.     

A systematic approach for extraction of phenolic compounds using parsley (Petroselinum crispum) flakes as a model substrate

The impact of extraction methodology and polarity of extraction solvents on the assay of phenolic compounds was investigated using parsley (Petroselinum crispum) flakes as a model substrate. This systematic study was undertaken to address substantial variations in the extraction procedures, solvents and conditions as described in the recent literature. Five different extraction procedures [shaking, vortex mixing, sonication, stirring and pressurized liquid extraction (PLE)] and three different solvents (methanol, ethanol and acetone), with five different solvent to water ratios per solvent, were used for extraction. Extracts were analyzed for phenolic content by high‐performance liquid chromatography and Folin–Ciocalteu assays. The yields of phenolic compounds extracted with a pressurized liquid extractor were comparable to or better than those of four classical extraction procedures. Optimum extraction efficiency with PLE was obtained when extractions were performed with four extraction cycles using ethanol–water (50:50, v/v). The amount of apiin (4,5,7‐trihydroxyflavone 7‐apiosylglucoside) and malonylapiin (apigenin malonylapiosylglucoside) isolated from parsley varied with the composition of extraction solvent. Apiin extractability was found to be a maximum when the solvent (ethanol, methanol or acetone) to water ratio was 30:70 (v/v), whereas higher amounts of malonylapiin were isolated with a reverse solvent to water ratio (70:30, v/v). Malonylapiin was not detected when parsley samples were extracted with organic solvent to water ratios of 10:90 (v/v) and 30:70 (v/v). Published in 2006 by John Wiley & Sons, Ltd     

Extraction of β-carotene produced from yeast Rhodosporidium sp. and its heat stability

This work determined the characteristics of β-carotene produced from Rhodosporidium sp. isolated from citrus fruits, and an effective extraction method was established. To extract β-carotene from the isolated Rhodosporidium sp., the cell walls were destroyed using dimethyl sulfoxide (DMSO) solution with and without the use of glass beads and a sonicator. Extracted β-carotene was identified by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) with a β-carotene standard. The yields of β-carotene extracted in DMSO, DMSO and glass beads, and DMSO with a sonicator were 3.371, 5.112, and 3.301 μg/mL, respectively. Isolated β-carotene was relatively heat stable, with 80% of the viable molecules remaining at 80°C.     

Carotenoids production from a newly isolated <Emphasis Type="Italic">Sporidiobolus pararoseus</Emphasis> strain by submerged fermentation

This work aimed at evaluating the total carotenoids production by a newly isolated Sporidiobolus pararoseus. Bioproduction was carried out in an orbital shaker, using 10% (w/v) of inoculum (25 °C, 180 rpm for 35 h), incubated for 120 h in a dark room. Liquid N₂ and dimethylsulphoxide (DMSO) were used for cell rupture, and carotenoids were extracted with a solution of acetone/methanol (7:3, v/v). Optimization of carotenoids bioproduction was achieved by experimental design technique. Initially, a Plackett–Burman design was used for the screening of the most important factors, after the statistical analysis, a complete second-order design was carried out to optimize the concentration of total carotenoids in a conventional medium. Maximum concentration of 856 μg/L of total carotenoids was obtained in a medium containing 60 g/L of glucose, 15 g/L of peptone, and 15 g/L of malt extract, 25 °C, initial pH 4.0 and 180 rpm. Fermentation kinetics showed that the maximum concentration of total carotenoids was reached after 102 h of fermentation and that carotenoids bioproduction was associated with cell growth.     

Major carotenoid composition of Brazilian Valencia orange juice: Identification and quantification by HPLC

The carotenoid composition of Brazilian Valencia orange juice was determined by open column chromatography (OCC) and high-performance liquid chromatography. Carotenoid pigments were extracted using acetone and saponified using 10% methanolic potassium hydroxide. Sixteen pigments were isolated by OCC and identified as α-carotene, ζ-carotene, β-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein-5,6-epoxide, violaxanthin, lutein, antheraxanthin, zeaxanthin, luteoxanthin A, luteoxanthin B, mutatoxanthin A, mutatoxanthin B, auroxanthin B and trollichrome B. Thirteen carotenoid pigments were separated using a ternary gradient (acetonitrile–methanol–ethyl acetate) elution on a C₁₈ reversed-phase column. Among these, violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, ζ-carotene, α-carotene, and β-carotene were quantified. The total carotenoid content was 12 ± 6.7 mg/l, and the major carotenoids were lutein (23%), β-cryptoxanthin (21%), and zeaxanthin (20%).     

Starch Derivatives of High Degree of Functionalization. 4. Homogeneous Tritylation of Starch and Subsequent Carboxymethylation

The synthesis of monomethoxytriphenylmethyl (MMtrityl) starch and the subsequent carboxymethylation of the 6‐O‐functionalized products were investigated. The trityIation both in N,N‐dimethylacetamide (DMA)/LiCl and in dimethyl sulfoxide (DMSO) occurred homogeneously. The highest degree of substitution of trityl groups (DS_TrityI) obtained after a single conversion step was 0.77 in both solvents. A complete functionalization of primary OH‐groups was achieved only with unsubstituted triphenylmethyl chloride in these reaction media. In case of monomethoxytriphenylmethyl chloride (MMTMC) as reagent an additional conversion step is necessary to synthesize products with a DS_TrityI = 1. The structure of the products was analyzed by FTIR‐ and ¹³C‐NMR spectroscopy. Subsequent carboxymethylation of the MMtrityl starch samples leads to products with a preferred functionalization of the unprotected secondary OH‐groups. After removal of the trityl moieties, the DS_CM and the distribution of carboxymethyl groups within the anhydroglucose unit was investigated by means of HPLC and ¹H‐NMR spectroscopy. The carboxymethylation was more effective at O‐2 than at O‐3. In case of ether products with DS_Trityl < 1 a partial substitution of the primary OH group took place as well.     

Multistage aqueous and non-aqueous extraction of bio-molecules from microalga Phaeodactylum tricornutum

A multi-step aqueous and non-aqueous extraction procedure was applied to recovery bio-molecules from Phaeodactylum tricornutum. The process include that physical pre-treatments (high voltage electrical discharges (HVED, 40 kV/cm, 1–8 ms, HVED samples) or high pressure homogenization (HPH, 1200 bar, 10 passes, P samples)) (1st step); aqueous extraction (2nd step); pigments extraction in ethanol (3rd step); and lipids extraction in CHCl₃/MeOH (4th step). The extractability of ionics, carbohydrates, proteins, pigments and lipids for untreated, HVED and P samples were evaluated. The results evidenced that HVED allowed selective extraction of water soluble ionic products at 1st and 2nd steps. The maximum ionic concentrations were found for HVED samples. However, P samples resulted in higher contents of extracted components as compared to HVED samples (≈1.5-fold of carbohydrates, ≈2.5-fold of proteins, ≈5-fold of carotenoids, and ≈3-fold of chlorophylls). Moreover, the non-aqueous extraction (3rd and 4th steps) allowed supplementary extraction of pigments and lipids.Industrial relevancePre-treatments by high voltage electrical discharge (HVED) or high pressure homogenization (HPH) allowed effective extraction of different bio-molecules from microalga biomass. The combination of HVED/HPH, aqueous and non-aqueous extraction allowed selective recovery of target molecules. The paper also presents a multistage extraction procedure allowed reducing the amount of toxic solvents, increasing the extraction efficiency, and reducing the energy consumption. These approaches opened the doors to high-efficiency recovery of valuable compounds from microalgae relevant for industrial application.     

Utilisation of orange by-products—orange peel carotenoids

Carotenoids were extracted from fresh orange peel with various solvents. Acetone was the most efficient of the solvents tested. Two successive extractions with acetone after an initial washing with either acetone or methanol were adequate to remove 89% of the total carotenoids. The extracts were concentrated, the carotenoids transferred to hexane and a crude pigment concentrate was obtained by hexane evaporation. Water washings prior to acetone extraction eliminated the solvent-solvent transfer to hexane. The extraction residue was used for pectin recovery. Carotenoid removal from the peel did not affect the yield and quality of the pectin.     

Optimization of microwave-assisted extraction of anthocyanins in red raspberries and identification of anthocyanin of extracts using high-performance liquid chromatography – mass spectrometry

Anthocyanins (Acys) are naturally occurring compounds that impart color to fruit, vegetables, and plants. The extraction of Acys from red raspberry (Rubus idaeus L. var. Heritage) by microwave-assisted process (MAP) was studied. A central composite rotate design (CCRD) was used to obtain the optimal conditions of microwave-assisted extraction (MAE), and the effects of operating conditions such as the ratio of solvents to materials, microwave power and extraction time on the extraction yield of Acys were studied through response surface methodology (RSM). The optimized conditions of MAE were ratio of solvents to materials 4:1 (ml/g), extraction time 12 min, and microwave power 366 W. Under these conditions 43.42 mg of Acys from 100 g of fresh fruits (T _Acy, expressed as cyanidin-3-glucoside), approximately 98.33% of the total red pigments, could be obtained by MAE. The Acys compositions of extracts were identified by high-performance liquid chromatography – mass spectrometry (HPLC-MS), 12 kinds of Acys had been detected and 8 kinds of Acys were characterized. Result indicated that cyanidin-3-sophoroside, cyanidin-3-(2 ^G -glucosylrutinoside), cyanidin-3-sambubioside, cyanidin-3-rutinoside, cyanidin-3-xylosylrutinoside, cyanidin-3-(2 ^G -glucosylrutinoside), and cyanidin-3-rutinoside were main components in extracts. In addition, in comparison with the conventional solvent extraction, MAE is more efficient and rapid to extract Acys from red raspberry, due to the strong disruption of fruit tissue structure under microwave irradiation, which had been observed with the scanning electron microscopepy (SEM). However, the Acys compositions in extracts by both the methods were similar, which were investigated using HPLC profile.     

Fractioned High Pressure Extraction of Anthocyanins from Elderberry (<Emphasis Type="Italic">Sambucus nigra</Emphasis> L.) Pomace

Fractionated high pressure extractions from dry and in natura elderberry pomace were performed in order to obtain anthocyanin rich extracts. Experiments were carried out using CO₂ supercritical fluid extraction followed by enhanced solvent extraction (ESE) with CO₂/EtOH–H₂O mixtures (1–100%, v/v), to obtain anthocyanin rich fractions in the second step, at 313 K and ~20 MPa. Higher extract yields, anthocyanin contents and antioxidant activities occurred by the presence of water, both in the raw material and in the solvent mixture. The CO₂ dissolved in the ESE solvent mixture favored either anthocyanin contents or antioxidant activities, which were not directly related. Comparing to the literature data for elderberries and grapes, these fractions had higher anthocyanins contents. From these results, an added economical value to this agroindustrial residue is proposed, using solvents and techniques “generally regarded as safe” in the food and pharmaceutical industries.