星期五, 二月 03, 2006 

Reference about Caroteniod separation

Extraction, separation and isolation of volatiles and dyes from Calendula officinalis L. and Aloysia triphylla (L'Her.) Britton by supercritical CO2 Journal of Essential Oil Research: JEOR, Sep/Oct 2003 by Crabas, Nicoletta, Marongiu, Bruno, Piras, Alessandra, Pivetta, Tiziana, Porcedda, Silvia
E. Stahl and D. Gerard, Solublitybehaviourand fractionation ofessential oils in dense carbon dioxide. Perfum. Flavor., 10(2) 29-37 (1985).
M. Richter, H. Sovova and J. Kucera, Solubility of monoterpenes in supercritical carbon dioxide. 2rd International Symposium on High Pressure Engineering. Erlangen (D), September 24-26 (1990).
E. Reverchon, Fractional separation of SFE extract from majorana leaves: mass transfer and optimization. J. Supercritical Fluids, 5, 256261 (1992).
Composition of Dalea formosa Oil Determined by Steam Distillation and Solid-Phase Microextraction, The
Journal of Essential Oil Research: JEOR, Nov/Dec 2005 by Lucero, Mary E, Estell, Rick E, Sedillo, Ruth L
TI: Improved separation method for highly purified lutein from Chlorella powder using jet mill and flash column chromatography on silica gel. AU: Shibata,-S; Ishihara,-C; Matsumoto,-K SO: Journal-of-Agricultural-and-Food-Chemistry. 2004; 52(20): 6283-6286 ; 16 ref. IS: 0021-8561 DT: Journal-Article PY: 2004
AB: An improved method for the separation of high-purified lutein from commercially available spray-dried Chlorella regularis powder (CP) using fine grinding by jet mill and flash column chromatography on a silica gel is described. Saponification and extraction of lutein were enhanced 2.3-2.9x in jet mill-treated CP (mean particle size = 20 mum) compared to untreated CP (mean particle size = 67 mum). The carotenoid extract was dissolved in ether-hexane (1:1 v/v) and subjected to flash column chromatography on silica gel. A mixture of alpha- and beta-carotene was eluted with hexane followed by elution with hexane-acetone-chloroform (7:2:1 v/v). Lutein (dark-orange band) was collected after the elution of an unknown colourless compound (detection based on UV absorbance). Purity of lutein in this fraction was >99% and the yield was 60%.

Preparative isolation and purification of lutein from the microalga Chlorella vulgaris by high-speed counter-current chromatography
Hua-Bin Lia, Feng Chen ian-You Zhangb, Fu-Quan Yangb and Guo-Qing Xub Received 8 August 2000; revised 20 September 2000; accepted 22 September 2000. Available online 12 December 2000.
Abstract
High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of lutein from microalgae. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of n-hexane–ethanol–water (4:3:1, v/v). Using the above solvent system, preparative HSCCC was successfully performed yielding lutein at 98% purity from 200 mg of the crude extract in a one-step separation.
Author Keywords: Lutein; Carotenoids; Vitamins
Title: Isolation of carotenoids from plant materials and dietary supplements by high-speed counter-current chromatography
Author(s): Aman R, Carle R, Conrad J, Beifuss U, Schieber A
Source: JOURNAL OF CHROMATOGRAPHY A 1074 (1-2): 99-105 MAY 13 2005
Abstract: Methods for the isolation of lipophilic pigments from crude extracts of plant materials (spinach and sweet corn) by high-speed counter-current chromatography (HSCCC) were developed. Particular attention was given to (all-E)-lutein and (all-E)-zeaxanthin. However, the concomitant pigments neoxanthin, violaxanthin and beta-carotene as well as chlorophylls a and b were also considered. Furthermore, for the first time dietary supplements containing lutein and zeaxanthin were also used as a source for the recovery of carotenoids. Due to their simple matrix (oily excipient in soft gelatine capsules), sample preparation was facilitated and consumption of solvents was minimized. The carotenoids were characterized by (HNMR)-H-1 spectroscopy, by LC/APcI-MS in the positive ionization mode, and by UV-vis spectroscopy. Data showed that the target compounds were of high purity (90-93%). Lutein and zeaxanthin may be used as reference substances for analytical purposes. (c) 2005 Elsevier B.V. All rights reserved.
Title: Isolation and purification of the bioactive carotenoid zeaxanthin from the microalga Microcystis aeruginosa by high-speed counter-current chromatography
Author(s): Chen F, Li HB, Wong RNS, Ji B, Jiang Y
Source: JOURNAL OF CHROMATOGRAPHY A 1064 (2): 183-186 FEB 4 2005
Abstract: High-speed counter-current chromatography was successfully applied for the first time to the isolation and purification of the bioactive carotenoid zeaxanthin from the cyanobacterium Microcystis aeruginosa. The crude zeaxanthin was obtained by extraction with organic solvents after the microalgal sample had been saponified. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v/v/v) was successfully performed yielding zeaxanthin at 96.2% purity from 150 mg of the crude extract in a one-step separation. The recovery of zeaxanthin was 91.4%. This was also the first report that zeaxanthin was successfully separated and purified from microalgae. (C) 2004 Published by Elsevier B.V.
Author Keywords: Microcystis aeruginosa; counter-current chromatography; preparative chromatography; zeaxanthin; carotenoids
Title: Separation and identification of lutein derivatives in processed foods
Author(s): Molnar P, Szabo Z, Osz E, Olah P, Toth G, Deli J
Source: CHROMATOGRAPHIA 60: S101-S105 Suppl. S, 2004
Abstract: In the present work, we show that in processed sorrel, the lutein (1) can convert to 3'-epilutein (2) - 3'-stereoisomer of lutein - by epimerisation reaction, as well as to anhydrolutein I (3) and II (4) by dehydration reaction. The newly formed anhydrolutein I (3) and II (4) were detected and identified by HPLC-DAD and HPLC-M5 techniques and co-chromatography with authentic samples. The compounds 1 and 2 were also isolated from cooked sorrel by column chromatography and characterized by NMR spectroscopy.
Author Keywords: column liquid chromatography-mass spectrometry; 3'-epilutein and anhydrolutein in sorrel; carotenoids
Title: Separation and determination of chloroplast pigments from spinach by thin-layer chromatography: a student laboratory experiment
Author(s): Sherma J, Fried B
Source: JPC-JOURNAL OF PLANAR CHROMATOGRAPHY-MODERN TLC 17 (4): 309-313 JUL-AUG 2004
Title: Extraction, separation and isolation of volatiles and dyes from Calendula officinalis L. and Aloysia triphylla (L'Her.) Britton by supercritical CO2
Author(s): Crabas N, Marongiu B, Piras A, Pivetta T, Porcedda S
Source: JOURNAL OF ESSENTIAL OIL RESEARCH 15 (5): 350-355 SEP-OCT 2003
Abstract: Isolation of volatile concentrate from the dried leaves of Aloysia triphylla (L'Herit.) Britton (lemon verbena) and the dried flowers of Calendula officinalis L. were obtained by supercritical extraction with CO2. To obtain a pure volatile extract devoid of cuticular waxes, the extraction products were fractionated in two separators operating in series. A good extraction process was obtained operating at 90 bar and 50degreesC in the extraction vessel, at 90 bar and at -5degreesC in the first separator and at a pressure between 20 and 15 bar and temperatures in the range (10-20degreesC) in the second one.
The composition of the volatile concentrate has been analyzed by GC/MS. The volatile concentrate of A. triphylla was found to contain: phytol(11.6%), spathulenol (7.1%), caryophyllene oxide (5.6%), methyl 9,12,15-octadecatrienoate (5.6%) and alpha-curcumene (4.6%). The volatile concentrate of C. officinalis was found to consist of: methyl hexadecanoate (23.8%), methyl linoleate (18.6%), methyl 9,12,15-octadecatrienoate (17.2%), methyl octadecanoate (4.8%), methyl tetradecanoate (4.6%), gamma-cadinene and cubenol (4.0%), delta-cadinene (3.2%), alpha-cadinol (1.8%) and oplopanone (1.3%). To complete the investigation, a comparison with the hydrodistilled oil has been carried out.
On the exhausted matrix a further extraction at higher pressure (320 bar) and 50degreesC with a single separator was performed for the extraction of lutein from Calendula flowers, the amount of lutein obtained was determined by spectrophotometric measurements.
Author Keywords: Aloysia triphylla; Verbenaceae; Calendula officinalis; Asteraceae; essential oil composition; supercritical CO2 extract composition; phytol; spathulenol; methyl hexadecanoate; methyl linoleate; methyl linolenate
Title: Application of CCC for the separation of lutein from a crude extract of marigold flower petals
Author(s): Wei Y, Zhang TY, Xu GQ, Ito Y
Source: JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 26 (9-10): 1659-1669 2003
Abstract: Analytical high-speed countercurrent chromatography (HSCCC) was used for the systematic selection and optimization of the two-phase solvent system to separate lutein from the crude extract of the marigold flower petal. Lutein was obtained by preparative HSCCC with a two-phase solvent system composed of n-heptane-chloroform-acetonitrile (10 : 3: 7, v/v/v). HPLC analyses of the lutein revealed that it was over 98.5% purity. The final identification was performed by UV-VIS absorption, H-1-NMR, and C-13-NMR Spectra.
Author Keywords: lutein; countercurrent chromatography; carotenoids; marigold flower petal
Title: A scheme for obtaining standards and HPLC quantification of leafy vegetable carotenoids
Author(s): Kimura M, Rodriguez-Amaya DB
Source: FOOD CHEMISTRY 78 (3): 389-398 AUG 2002
Abstract: Reliability of data obtained by HPLC directly depends on the accuracy of the calibration. A major difficulty is obtaining and maintaining pure standards. This work presents a strategy for isolating standards by open column chromatography and quantification by HPLC, using leafy vegetables as examples. The purity of standards evaluated by HPLC was 91-97% for neoxanthin, 95-98% for violaxanthin, 97-100% for lactucaxanthin, 92-96% for lutein and 90-97% for beta-carotene. Calculation procedures were also evaluated, demonstrating that results obtained with one-point recalibration, straight-line equation (until 30 days after construction of the full calibration curves) and response factors relative to beta-carotene were similar (CVs of 1.6-4.0%), being well below between-sample lot natural variation (CVs of 6.1-42.5%). The scheme proposed is relatively low-cost, provides a constant supply of carotenoid standards, including those unavailable commercially, and high sample throughput. (C) 2002 Elsevier Science Ltd. All rights reserved.
Author Keywords: carotenoids; HPLC; analysis; standards
Title: A fast, reliable and low-cost saponification protocol for analysis of carotenoids in vegetables
Author(s): Granado F, Olmedilla B, Gil-Martinez E, Blanco I
Source: JOURNAL OF FOOD COMPOSITION AND ANALYSIS 14 (5): 479-489 OCT 2001
Abstract: Carotenoids are mainly present in vegetables and fruits and display several important biological actions (provitamin in A, antioxidant and anticarcinogenic activities and immunomoduiation). To assess the intake of carotenoids and their potential health benefits in humans, it is necessary to have reliable information about carotenoid content in food. Saponification is commonly used during analysis of carotenoids in vegetables and fruits to release esterified xanthophylls and to eliminate interfering substances. It is a time-consuming step and involves the use of considerable volumes of solvents in the partition. To reduce the time and costs of the saponification process, we compare the reliability of a "shortcut" (small volumes, vortex 3 min, 20% potassium hydroxide, hexane/methylene chloride extraction) with that of a "standard" protocol (stirring 30 min, 5% potassium hydroxide, petroleum ether/diethyl ether extraction twice plus washings). Accuracy and precision were assessed according to studies of certified reference materials for carotenoid analysis in foods. For lutein, zeaxanthin, chi-cryptoxanthin, beta-cryptoxanthin, lycopene, chi-carotene and beta-carotene, the "shortcut" showed within-day and between-day variability similar to or lower than that of the "standard" protocol, comparable deviation from the assigned values and higher recovery for carotenes, especially for lycopene. Advantages of the "shortcut" include considerably reduced time, higher overall recoveries and lower solvent expenditure, resulting in a reduction in time and total cost of up to 80-90% without loss of accuracy or precision. (C) 2001 Academic Press.
Author Keywords: carotenoids; saponification; xanthophylls; vegetables; fruits
Title: Improved extraction procedure for carotenoids from human milk
Author(s): Schweigert FJ, Hurtienne A, Bathe K
Source: INTERNATIONAL JOURNAL FOR VITAMIN AND NUTRITION RESEARCH 70 (3): 79-83 MAY 2000
Abstract: An improves method for the extraction of the major carotenoids from human milk is described. Carotenoids were extracted from milk first with ethanol and n-hexane. Then, polar xanthophylls were extracted from n-hexane into ethanol/water. The remaining n-hexane was evaporated, the residue combined with the ethanolic milk fraction and the mixture briefly saponified. Carotenoids were extracted from the hydrolysate with n-hexane, combined with the polar xanthophylls from the non-saponified ethanol/water-extract and separated by HPLC. Using this method we were able to significantly improve the recovery of xanthophylls such as lutein and zeaxanthin from human milk. The recovery rate of all carotenoids was > 90%. This method might not only be of value for milk but should be especially useful in the extraction of carotenoids from human tissues such as the adipose tissue.
Author Keywords: xanthophylls; saponification; milk; human; method
Title: Processing and stability of carotenoid powder from carrot pulp waste
Author(s): Chen BH, Tang YC
Source: JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 46 (6): 2312-2318 JUN 1998
Abstract: Carrot pulp waste was used as raw material for processing carotenoid powder by spray-drying, and the stability of carotenoid in the powder was studied under light and dark storage at various temperatures. The various carotenoids were analyzed by HPLC with photodiode array detection. Results showed that the most appropriate condition for processing carotenoid powder by spray-drying consists of 15% solid content of feed, with inlet air temperature of 135-145 degrees C and outlet air temperature of 90-100 degrees C. The total amount of all-trans plus cis forms of lutein, alpha-carotene, or beta-carotene in the carotenoid powder decreased with increasing storage time and temperature, and the degradation rate of each fits the first-order model. The major cis isomers formed in the dark were 13-cis-alpha-carotene and 13-cis-beta-carotene, whereas 9-cis isomers of both alpha- and beta-carotene predominated under light. A high correlation was also observed between color changes and carotenoid content.
Author Keywords: carotenoid powder; carrot pulp waste; spray-drying; carotenoid stability
Title: Preparation of lutein from Marigold flowers and esterification to their myristates
Author(s): Subagio A, Morita N
Source: ANALYTICAL SCIENCES 13 (6): 1025-1028 DEC 1997
Author Keywords: carotenoid fatty acid ester; lutein; lutein monomyristate; lutein dimyristate; Marigold flower
Title: Effects of enzymatic treatments of marigold flowers on lutein isomeric profiles
Author(s): DelgadoVargas F, ParedesLopez O
Source: JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 45 (4): 1097-1102 APR 1997
Abstract: Reversed-phase C-30 HPLC was applied to study the identity of lutein isomers and to monitor the effects of solids content and elimination of water-soluble substances on the isomeric carotenoid profiles of marigold (Tagetes erecta) samples treated with enzymes. The tentative identity of four lutein isomers present in saponified marigold extracts was confirmed. Enzymatic treatment on a 5% solids slurry produced the marigold meal with the highest all-trans-lutein content [25.1 g/kg dry weight(dw)]. We did not observe variations in the distribution in percentage of lutein isomers due to enzymatic treatment; the elimination of water solubles had a significant but small effect on such variations. The solids content was the principal factor that affected the carotenoid profiles. An analysis of the distribution showed that 15% solids gave the highest all-trans-lutein percentage in treated meals. Interestingly, with 20% solids both the degradation of lutein and the percentage of all-trans-zeaxanthin were the highest.
Author Keywords: lutein; carotenoids; pigments; marigold; Tagetes
Carotenoid and Carotenoid Ester Composition in Mango Fruit As Influenced by Processing MethodM. Pilar Cano and Begona de AncosJ. Agric. Food Chem.; 1994; 42(12) pp 2737 - 2742;
CRAFT NE, WISE SA, SOARES JHOPTIMIZATION OF AN ISOCRATIC HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SEPARATION OF CAROTENOIDS
Abstract
Using a polymeric C18 high-performance liquid chromatographic (HPLC) column, which demonstrated excellent separation selectivity toward carotenoid compounds in an earlier column evaluation, the effects of mobile phase modifier, modifier concentration, and column temperature were investigated. A seven-component carotenoid mixture was used to monitor changes in separation selectivity in response to variations in HPLC conditions. Both acetonitrile and tetrahydrofuran (THF) improved the resolution of echinenone and α-carotene; THF was selected for use as a modifier due to its solvating properties. At concentrations greater than 6% THF, the resolution of lutein and zeaxanthin deteriorated significantly. Temperature was varied from 15 to 35°C in 5°C increments. Resolution of lutein/zeaxanthin and β-cartone/lycopene were better at lower temperatures while echinenone/α-carotene separation improved as temperature increased. An acceptable separation of all seven carotenoids was achieved at 20°C using 5% THF as a mobile phase modifier. Method applicability is demonstrated for serum and food carotenoids.
JOURNAL OF CHROMATOGRAPHY 589 (1-2): 171-176 JAN 10 1992
HPLC separation of chlorophyll and carotenoid pigments of four kiwifruit cultivarsM. Pilar Cano.J. Agric. Food Chem.; 1991; 39(10) pp 1786 - 1791;
Shi J, Le Maguer MLycopene in tomatoes: Chemical and physical properties affected by food processing CRITICAL REVIEWS IN BIOTECHNOLOGY 20 (4): 293-334 2000
Ibanez E, Lopez-Sebastian S, Tabera J, et al.Separation of carotenoids by subcritical fluid chromatography with coated, packed capillary columns and neat carbon dioxide JOURNAL OF CHROMATOGRAPHY A 823 (1-2): 313-319 OCT 9 1998
Taungbodhitham AK, Jones GP, Wahlqvist ML, et al.Evaluation of extraction method for the analysis of carotenoids in fruits and vegetables FOOD CHEMISTRY 63 (4): 577-584 DEC 1998
BISHOP NI, URBIG T, SENGER HCOMPLETE SEPARATION OF THE BETA,EPSILON-CAROTENOID AND BETA,BETA-CAROTENOID BIOSYNTHETIC PATHWAYS BY A UNIQUE MUTATION OF THE LYCOPENE CYCLASE IN THE GREEN-ALGA, SCENEDESMUS-OBLIQUUS FEBS LETTERS 367 (2): 158-162 JUN 26 1995
STAHL W, SIES HSEPARATION OF GEOMETRICAL-ISOMERS OF BETA-CAROTENE AND LYCOPENE METHODS IN ENZYMOLOGY 234: 388-400 1994
GOODRICH J, PARKER C, PHELPS RTHE MICROSCALE SEPARATION OF LYCOPENE AND BETA-CAROTENE FROM TOMATO PASTE JOURNAL OF CHEMICAL EDUCATION 70 (6): A158-A158 JUN 1993
HL W, SUNDQUIST AR, HANUSCH M, et al.SEPARATION OF BETA-CAROTENE AND LYCOPENE GEOMETRICAL-ISOMERS IN BIOLOGICAL SAMPLES CLINICAL CHEMISTRY 39 (5): 810-814 MAY 1993
RONMAN PIMPROVEMENTS IN THE SEPARATION OF BETA-CAROTENE AND LYCOPENE BY COLUMN CHROMATOGRAPHY JOURNAL OF CHEMICAL EDUCATION 62 (6): 540-540 1985 Montero O, Macias-Sanchez MD, Lama CM, et al.Supercritical CO2 extraction of beta-carotene from a marine strain of the cyanobacterium Synechococcus species JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 53 (25): 9701-9707 DEC 14 2005
Bhosale P, Bernstein PSbeta-carotene production by Flavobaeterium multivorum in the presence of inorganic salts and urea
Abstract Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml–1 h–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.
Keywords Flavobacterium multivorum - Zeaxanthin - -Carotene - -Cryptoxanthin - Response surface methodology
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY 31 (12): 565-571 DEC 2004
Park PK, Cho DH, Kim EY, et al.Optimization of carotenoid production by Rhodotorula glutinis using statistical experimental design
Abstract A two-step optimization strategy of statistical experimental design was employed to enhance carotenoid production from sugar cane molasses (SCM) in the yeast Rhodotorula glutinis. In the first step, a fractional factorial design was used to evaluate the impact of five fermentation factors (pH and concentrations of SCM, urea, KH2PO4, and NaCl). The results revealed that three factors (concentrations of SCM, urea, and KH2PO4) had a significant influence on biomass and carotenoid production. A face-centered central composite design was then used in the second step to optimize the three significant factors to further enhance the biomass yield and carotenoid production. Through this two-step optimization strategy, the carotenoid concentration could be increased from an average of 1.39 mg/l to an average of 3.46 mg/l, representing a 2.5-fold carotenoid production enhancement.WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 21 (4): 429-434 JUN 2005
Tinoi J, Rakariyatham N, Deming RLSimplex optimization of carotenoid production by Rhodotorula glutinis using hydrolyzed mung bean waste flour as substrate PROCESS BIOCHEMISTRY 40 (7): 2551-2557 JUN 2005
You LL, Abdullah MA, Quek SY, et al.Optimization of carotene recovery from hydrolyzed palm olein in adsorption column chromatography using response surface methodology JOURNAL OF FOOD LIPIDS 11 (1): 45-55 APR 2004
Hartono R, Mansoori GA, Suwono APrediction of solubility of biomolecules in supercritical solvents CHEMICAL ENGINEERING SCIENCE 56 (24): 6949-6958 DEC 2001
Careri M, Furlattini L, Mangia A, et al.Supercritical fluid extraction for liquid chromatographic determination of carotenoids in Spirulina Pacifica algae: a chemometric approach JOURNAL OF CHROMATOGRAPHY A 912 (1): 61-71 MAR 30 2001
Lang QY, Wai CMSupercritical fluid extraction in herbal and natural product studies - a practical review TALANTA 53 (4): 771-782 JAN 5 2001
Lesellier E, Tchapla ASeparation of vegetable oil triglycerides by subcritical fluid chromatography with octadecyl packed columns and CO2/modifier mobile phase CHROMATOGRAPHIA 51 (11-12): 688-694 JUN 2000 Times Cited: 5
Kalampoukas G, Dervakos GAProcess optimization for clean manufacturing: Supercritical fluid extraction for B-carotene production COMPUTERS & CHEMICAL ENGINEERING 20: S1383-S1388 Suppl. B 1996
Sanal IS, Bayraktar E, Mehmetoglu UU, et al.Determination of optimum conditions for SC-(CO2 plus ethanol) extraction of beta-carotene from apricot pomace using response surface methodology JOURNAL OF SUPERCRITICAL FLUIDS 34 (3): 331-338 JUL 2005
Sanagi MM, See HH, Ibrahim WAW, et al.Determination of carotene, tocopherols and tocotrienols in residue oil from palm pressed fiber using pressurized liquid extraction-normal phase liquid chromatography ANALYTICA CHIMICA ACTA 538 (1-2): 71-76 MAY 4 2005
Jaramillo-Flores ME, Lugo-Martinez JJ, Ramirez-SanJuan E, et al.Effect of sodium chloride, acetic acid, and enzymes on carotene extraction in carrots (Daucus carota L.) JOURNAL OF FOOD SCIENCE 70 (2): S136-S142 MAR 2005
Hejazi MA, Kleinegris D, Wijffels RHMechanism of extraction of beta-carotene from microalga Dunaliellea salina in two-phase bioreactors BIOTECHNOLOGY AND BIOENGINEERING 88 (5): 593-600 DEC 5 2004
Doker O, Salgin U, Sanal I, et al.Modeling of extraction of beta-carotene from apricot bagasse using supercritical CO2 in packed bed extractor JOURNAL OF SUPERCRITICAL FLUIDS 28 (1): 11-19 JAN 2004
Sabio E, Lozano M, de Espinosa VM, et al.Lycopene and beta-carotene extraction from tomato processing waste using supercritical CO2 INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH 42 (25): 6641-6646 DEC 10 2003
Gimeno E, Castellote AI, Lamuela-Raventos RM, et al.The effects of harvest and extraction methods on the antioxidant content (phenolics, alpha-tocopherol, and beta-carotene) in virgin olive oil FOOD CHEMISTRY 78 (2): 207-211 AUG 2002
Baharin BS, Latip RA, Man YBC, et al.The effect of carotene extraction system on crude palm oil quality, carotene composition, and carotene stability during storage JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY 78 (8): 851-855 AUG 2001
Amarowicz R, Karamac M, Chavan UInfluence of the extraction procedure on the antioxidative activity of lentil seed extracts in a beta-carotene-linoleate model system GRASAS Y ACEITES 52 (2): 89-93 MAR-APR 2001
Chan KW, Baharin BS, Man YBCAdsorption isotherm studies of palm,carotene extraction by synthetic polymer adsorbent JOURNAL OF FOOD LIPIDS 7 (2): 127-141 JUN 2000
Ye L, Landen WO, Eitenmiller RRSimplified extraction procedure and HPLC determination for total vitamin E and beta-carotene of reduced-fat mayonnaise JOURNAL OF FOOD SCIENCE 66 (1): 78-82 JAN-FEB 2001
Roukas T, Mantzouridou FAn improved method for extraction of beta-carotene from Blakeslea trispora
Abstract:An improved method for the extraction of β-carotene from Blakeslea trispora is described. The fermentation broth was steamed at 121°C for 15 min, and the liquid was centrifuged at 5000g for 20 min. β-Carotene was removed from the biomass by extraction with absolute ethanol at a ratio of 1:100 at 30°C for 2 h in a rotary shaker incubator at 300 rpm. The carotenoid pigment was completely removed from the cells after three repeated extractions. The removal of β-carotene from B. trispora was higher during the first stage (75%) whereas in the other stages it was very slow.APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 90 (1): 37-45 JAN 2001
Latip RA, Baharin BS, Man YBC, et al.Effect of adsorption and solvent extraction process on the percentage of carotene extracted from crude palm oil JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY 78 (1): 83-87 JAN 2001
Ray KS, Chheda M, Mukhopadhyay MPerformance of conventional and super critical fluid extraction methods for carotene recovery from non-edible leaves JOURNAL OF FOOD SCIENCE AND TECHNOLOGY-MYSORE 37 (5): 514-516 SEP-OCT 2000
Cocero MJ, Gonzalez S, Perez S, et al.Supercritical extraction of unsaturated products. Degradation of beta-carotene in supercritical extraction processes JOURNAL OF SUPERCRITICAL FLUIDS 19 (1): 39-44 NOV 15 2000
Baysal T, Ersus S, Starmans DAJSupercritical CO2 extraction of beta-carotene and lycopene from tomato paste waste JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 48 (11): 5507-5511 NOV 2000
de Franca LF, Meireles MAAModeling the extraction of carotene and lipids from pressed palm oil (Elaes guineensis) fibers using supercritical CO2 JOURNAL OF SUPERCRITICAL FLUIDS 18 (1): 35-47 AUG 10 2000
Cadoni E, De Giorgi MR, Medda E, et al.Supercritical CO2 extraction of lycopene and beta-carotene from ripe tomatoes DYES AND PIGMENTS 44 (1): 27-32 DEC 2 1999
Weathers RM, Beckholt DA, Lavella AL, et al.Comparison of acetals as in situ modifiers for the supercritical fluid extraction of beta-carotene from paprika with carbon dioxide JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 22 (2): 241-252 1999
Subra P, Castellani S, Jestin P, et al.Extraction of beta-carotene with supercritical fluids - Experiments and modelling JOURNAL OF SUPERCRITICAL FLUIDS 12 (3): 261-269 JUL 1 1998
Chandra A, Nair MGSupercritical fluid carbon dioxide extraction of alpha- and beta-carotene from carrot (Daucus carota L.)
Abstract Carrots (Daucus carota L. var. Caro Pride) were extracted with supercritical fluid carbon dioxide (SFOC2) under various combinations of pressures and solvent modifiers at 40°C and analysed for their - and -carotene content by high pressure liquid chromatography. The SFCO2 extraction at 40°C and 60.6 MPa with 5% chloroform as modifier afforded 111.16 and 148.32 g of - and -carotene per gram of dried carrot, respectively. Also, this method extracted 92.70% of the total carotenoids present in the dried carrots when compared to the solvent extraction using chloroform (100%)PHYTOCHEMICAL ANALYSIS 8 (5): 244-246 SEP-OCT 1997
Lenfant C, Thyrion FCExtraction of carotenoids from palm oil .1. Physical and chemical properties of beta-carotene OCL-OLEAGINEUX CORPS GRAS LIPIDES 3 (3): 220-226 MAY-JUN 1996
Kalampoukas G, Dervakos GAProcess optimization for clean manufacturing: Supercritical fluid extraction for B-carotene production COMPUTERS & CHEMICAL ENGINEERING 20: S1383-S1388 Suppl. B 1996
MARSILI R, CALLAHAN DCOMPARISON OF A LIQUID SOLVENT-EXTRACTION TECHNIQUE AND SUPERCRITICAL-FLUID EXTRACTION FOR THE DETERMINATION OF ALPHA-CAROTENE AND BETA-CAROTENE IN VEGETABLES JOURNAL OF CHROMATOGRAPHIC SCIENCE 31 (10): 422-428 OCT 1993
SPANOS GA, CHEN H, SCHWARTZ SJSUPERCRITICAL CO2 EXTRACTION OF BETA-CAROTENE FROM SWEET-POTATOES JOURNAL OF FOOD SCIENCE 58 (4): 817-820 JUL-AUG 1993
SADLER G, DAVIS J, DEZMAN DRAPID EXTRACTION OF LYCOPENE AND BETA-CAROTENE FROM RECONSTITUTED TOMATO PASTE AND PINK GRAPEFRUIT HOMOGENATES JOURNAL OF FOOD SCIENCE 55 (5): 1460-1461 SEP-OCT 1990
FAVATI F, KING JW, FRIEDRICH JP, et al.SUPERCRITICAL CO2 EXTRACTION OF CAROTENE AND LUTEIN FROM LEAF PROTEIN-CONCENTRATES JOURNAL OF FOOD SCIENCE 53 (5): 1532-1536 SEP-OCT 1988
LAW KN, LO SN, KORAN ZUTILIZATION OF SPRUCE FOLIAGE - EXTRACTION OF PROTEIN AND CHLOROPHYLL-CAROTENE WOOD SCIENCE 11 (2): 91-96 1978
FISHER JF, ROUSEFF RLSOLID-PHASE EXTRACTION AND HPLC DETERMINATION OF BETA-CRYPTOXANTHIN AND ALPHA-CAROTENE AND BETA-CAROTENE IN ORANGE JUICE JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 34 (6): 985-989 NOV-DEC 1986
Lu YJ, Yao HY, Gong YS, et al.Effectes of enzymatic treatment of corn gluten meal on lutein and zeaxanthin extraction INTERNATIONAL JOURNAL OF FOOD PROPERTIES 8 (1): 161-169 2005
Rao RLutein diesters extract from super critical fluid extraction process AGRO FOOD INDUSTRY HI-TECH 14 (2): 19-22 MAR-APR 2003
Li HB, Jiang Y, Chen FIsolation and purification of lutein from the microalga Chlorella vulgaris by extraction after saponification JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 50 (5): 1070-1072 FEB 27 2002
Shi XM, Chen FProduction and vapid extraction of lutein and the other lipid-soluble pigments from Chlorella protothecoides grown under heterotrophic and mixotrophic conditions NAHRUNG-FOOD 43 (2): 109-113 APR 1999
Lavecchia R, Zuorro AEnzyme-mediated extraction of lycopene from tomato skins. ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 227: U35-U35 047-AGFD Part 1 MAR 28 2004
Periago MJ, Rincon F, Aguera MD, et al.Mixture approach for optimizing lycopene extraction from tomato and tomato products JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 52 (19): 5796-5802 SEP 22 2004
Yang SX, Shi WR, Zeng JModelling the supercritical fluid extraction of lycopene from tomato paste waste using neuro-fuzzy approaches LECTURE NOTES IN COMPUTER SCIENCE 3174: 880-885 2004
Vasapollo G, Longo L, Rescio L, et al.Innovative supercritical CO2 extraction of lycopene from tomato in the presence of vegetable oil as co-solvent JOURNAL OF SUPERCRITICAL FLUIDS 29 (1-2): 87-96 APR 2004
Gomez-Prieto MS, Caja MM, Herraiz M, et al.Supercritical fluid extraction of all-trans-lycopene from tomato JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 51 (1): 3-7 JAN 1 2003
Rozzi NL, Singh RK, Vierling RA, et al.Supercritical fluid extraction of lycopene from tomato processing byproducts JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 50 (9): 2638-2643 APR 24 2002
Cadoni E, De Giorgi MR, Medda E, et al.Supercritical CO2 extraction of lycopene and beta-carotene from ripe tomatoes DYES AND PIGMENTS 44 (1): 27-32 DEC 2 1999
Inakuma T, Yasumoto M, Koguchi M, et al.Effect of drying methods on extraction of lycopene in tomato skin with supercritical carbon dioxide JOURNAL OF THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY-NIPPON SHOKUHIN KAGAKU KOGAKU KAISHI 45 (12): 740-743 1998
SADLER G, DAVIS J, DEZMAN DRAPID EXTRACTION OF LYCOPENE AND BETA-CAROTENE FROM RECONSTITUTED TOMATO PASTE AND PINK GRAPEFRUIT HOMOGENATES JOURNAL OF FOOD SCIENCE 55 (5): 1460-1461 SEP-OCT 1990
ADSULE PG, DAN ASIMPLIFIED EXTRACTION PROCEDURE IN THE RAPID SPECTROPHOTOMETRIC METHOD FOR LYCOPENE ESTIMATION IN TOMATO JOURNAL OF FOOD SCIENCE AND TECHNOLOGY-MYSORE 16 (5): 216-216 1979

6:13 上午 | |

 

Aqueous Enzymatic Extraction of Oil from Rapeseeds

Background:
Vegetable oils and animal fats are important ingredients for several different foodstuffs and non-food products. About 80% of the total market of oils and fats is of vegetable origin [1]. Rapeseed contains oil (40-46%) and protein (20-30%) of high quality, but also glucosinolates which can be critical for both oil and protein quality. Especially if too high concentrations of these compounds or their degradation products are present in the oil and protein products. Novel processing methods of rapeseed have therefore attracted appreciable attention [2].

Oil from rape seed is traditionally produced by a combined process using pressing followed by extraction with organic solvents, like hexane. These organic solvents are recycled. This process creates in principle two products, the oil and the low-valued meal, that is used as feed ingredient or as fertilizer. The content of glucosinolates, the too high content of cellulose and tannins, aromatic cholinesters, and phytic acid in the conventional rapeseed meal still present some nutritional disadvantages. It is only usable for cows. The use of solvents presents a constant danger of explosion or fire risks and pollution [3,4]. In some countries (United States) the building of new factories using hexane to extract the oil is prohibited [5].

5:57 上午 | |

 

Cellulase Enzyme Research

Cellulase Enzyme Research


The goal is to reduce the cost of using cellulase enzymes in the bioethanol process by employing cutting-edge and efficient biochemical technologies. Our current estimate for cellulase ranges from 30 to 50 cents per gallon of ethanol produced. The objective is to reduce cellulase cost to less than 5 cents per gallon of ethanol. This requires a tenfold increase in specific activity or production efficiency or some combination thereof. Nearer-term goals include a threefold increase in cellulase-specific activity (relative to the Trichoderma reesei system) by FY 2005.

Dilute acid pretreatment of hard woods tends to yield residual biomass that is high in native cellulose and very low in hemicellulosic sugars. In the early 1990s, NREL researchers determined that a 90:9:1 mixture of a cellobiohydrolase from T. reesei (CBH I), a thermal-tolerant endoglucanase from A. cellulolyticus (EI), and a ß-D-glucosidase was capable of nearing the performance observed for a leading commercial T. reesei preparation at comparable protein loading (i.e., end-point saccharification of cellulose in pretreated yellow poplar after 120 hours). This discovery was an important breakthrough because the option to engineer cellulase systems for specific pretreated biomass feedstocks became apparent for the first time.

Subcontract:
Develop and Test Improved T. fusca Cellulases
Cornell University (D. Wilson) is evaluating new cellulase mixtures and their application to biomass as well as working to improve T. fusca E3 and E5 by site-directed-mutagenesis.
Protein Engineering

Work at NREL in FY 1997 to 1998, with collaboration from the University of Arkansas (J. Sakon), presented another breakthrough in the cellulase biochemistry field. We showed that the performance (measured as maximum final percent saccharification of biomass) of this ternary system was improved by 13% following site-directed modification of one active site amino acid residue identified from a high resolution X-ray crystallographic structure of EI. We are now pursuing other promising classes of EI mutations — those that modify the chemistry of the biomass interactive surface.

Subcontracts:
Provide High Resolution X-ray Structures
The University of Arkansas (J. Sakon) is working to support site-directed-mutagenesis studies of Acidothermus EI and T. reesei CBH I.

Molecular Modeling of the Interaction of Cellulose with Cellulases and Catalysts
Cornell University (J. Brady) is working to apply molecular mechanics modeling to enhance our understanding of cellulase action. This information is being used to design protein engineering experiments at NREL and elsewhere.

Advanced Cellulase Performance Assays
The analytical membrane-reactor assay for cellulases developed at NREL, the diafiltration saccharification assay (DSA), produces very precise and detailed progress curves for the enzymatic saccharification of cellulosic materials under conditions that mimic those of simultaneous saccharification and fermentation. Recently, extensive enzyme-loading studies have been carried out, using the DSA, on the kinetics of digestion of dilute-acid-pretreated yellow poplar by three different T. reesei cellulase preparations. Analysis of the combined results demonstrates that an approach utilizing "time-to-target" kinetics and plots of the resulting "reciprocal-time-to-target" values versus a function of cellulase loading show highly linear relationships. These linear relationships constitute a convenient "shorthand" method of describing the performance of a given cellulase preparation over a wide range of loading and reaction times.

Subcontract:
Design and Test Improved Assays for Endoglucanases and Exoglucanases
The University of California at Davis (S. Shoemaker) is working to better understand the mechanisms of cellulase action. Methods for evaluating the performance of endoglucanases and exoglucanases acting on soluble and insoluble substrates will be developed.

Trichoderma reesei Proteonomics
For more complex biomass feedstocks, understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. We presume that the following enzymes are important in hydrolyzing biomass: ß-1,4-endoglucanases (EG I, EG II, EG III, and EG V); ß-1,4-cellobiohydrolases (CBH I & CBH II); xylanases (XYN I & XYN II); ß-glucosidase; a-L-arabinofuranosidase; acetyl xylan esterase; ß-mannanase; and a-glucuronidase. At NREL, these enzymes are being fingerprinted from T. reesei broth by 2-D gel electrophoresis and confirmed by direct peptide sequence analysis. The compositions of variably induced cellulase preparations from T.reesei are also being compared by this method and related to the overall activity of these cellulase preparations using the diafiltration saccharification assay.

Recent Publications, Reports, and Presentations
General
Himmel, M.E.; Adney, W.S.; Baker, J.O.; Elander, R.; McMillan, J.D.; Nieves, R.A.; Sheehan, J.; Thomas, S.R.; Vinzant, T.B.; Zhang, M. (1997). "Advanced Bioethanol Production Technologies: A Perspective." Woodward, J.; Saha, B., eds. Fuels and Chemicals from Biomass, ACS Series 666, Washington, DC: American Chemical Society; pp. 2-45.
Himmel, M.E.; Baker, J.O.; Saddler, J., eds. (2001). "Glycosyl Hydrolases for Biomass Conversion." ACS Symposium Series 769, Washington, DC: American Chemical Society: Distributed by Oxford University Press.
Sheehan, J.; Himmel, M.E. (1999). "Enzymes, Energy, and the Environment: Cellulase Development in the Emerging Bioethanol Industry." Biotechnology Progress (15:3); pp. 817-827.

Engineered Cellulase Mixtures
Baker, J.O.; Adney, W.S.; Nieves, R.A.; Thomas, S.R.; Himmel, M.E. (1995). "Synergism in Binary Mixtures of Bacterial and Fungal Cellulases: Endo/Exo, Exo/Exo, and Endo/Endo Interactions." Saddler, J.N.; Penner, M.H. eds. Enzymatic Degradation of Insoluble Polysaccharides, ACS Series 618, Washington, DC: American Chemical Society; pp. 113-141.
Baker, J.O.; Ehrman, C.I.; Adney, W.S.; Thomas, S.R.; Himmel, M.E. (1998) "Hydrolysis of Cellulose Using Ternary Mixtures of Purified Cellulases." Appl. Biochem. Biotechnol. (70:72); pp. 395-403.
Baker, J.O.; King, M.R.; Adney, W.S.; Decker, S.R.; Vinzant, T.B.; Lantz, S.L.; Nieves, R.E.; Thomas, S.R.; Li, L.-C.; Cosgrove, D.J.; Himmel, M.E. (1999). "Investigation of the Cell Wall Loosening Protein Expansion as a Possible Additive in the Enzymatic Saccharification of Lignocellulosic Biomass," Applied Biochemistry and Biotechnology, In Press.
Thomas, S.R.; Laymon, R.A.; Chou, Y.C.; Tucker, M.P.; Vinzant, T.B.; Adney, W.S.; Baker, J.O.; Nieves, R.A.; Mielenz, J.R.; Himmel, M.E. (1995). "Initial Approaches to Artificial Cellulase Systems for Conversion of Biomass to Ethanol." Saddler, J.N.; Penner, M.H., eds. Enzymatic Degradation of Insoluble Polysaccharides, ACS Series 618, Washington, DC: American Chemical Society; pp. 208-236.

Protein Engineering
Godbole, S.; Decker, S.R.; Nieves, R.A.; Adney, W.S.; Vinzant, T.B.; Baker, J.O.; Thomas, S.R.; Himmel, M.E. (1999) "Cloning and Expression of Trichoderma reesei CBH I in Pichia pastoris," Biotechnology Progress (15:3); pp. 828-833.
Himmel, M.E.; Adney, W.S.; Baker, J.O.; Nieves, R.A.; Thomas, S.R. (1996) "Cellulases: Structure, Function, and Application," Wyman, C.E., ed., Handbook on Bioethanol, Washington, DC: Taylor & Francis; pp. 143-161.
Karplus, P.A.; Sakon, J.; Adney, W.S.; Baker, J.O.; Thomas, S.R. (1997) "Polysaccharide Hydrolase Folds: Diversity of Structure and Convergence of Function," Appl. Biochem. Biotechnol. (63:65); pp. 315-326.
Palma, R.; Himmel, M.E.; Liang, G.; Brady, J. (2000) "Molecular Mechanics Studies of Cellulases," Himmel, M.E.; Baker, J.O.; Saddler, J., eds. Glycosyl Hydrolases for Biomass Conversion, ACS Series 769, Washington, DC: American Chemical Society, In press.
Sakon, J.; Adney, W.; Himmel, M; Thomas, S.; Karplus, P. (1996) "Crystal Structure of Thermostable Family 5 Endocellulase EI from Acidothermus cellulolyticus in Complex with Cellotetraose," Biochemistry (35); pp. 10648-10660.
Advanced Cellulase Performance Assays
Baker, J.O.; Vinzant, T.B.; Ehrman, C.I.; Adney, W.S.; Himmel, M.E. (1997) "A Membrane-Reactor Saccharification Assay to Evaluate the Performance of Cellulases Under Simulated SSF Conditions," Appl. Biochem. Biotechnol., (63:65); pp. 585-595.
Vinzant, T.B.; Ehrman, C.I.; Himmel, M.E. (1997) "SSF of Pretreated Hardwoods: Effect of Native Lignin Content." Appl. Biochem. Biotechnol., (62:1); pp. 97-102.

5:44 上午 | |

 

Pretreatment Technology Evaluation

Pretreatment Technology Evaluation

In general, pretreatment processes produce a solid pretreated biomass residue that is more amenable to enzymatic hydrolysis by cellulases and related enzymes than native biomass. Many pretreatment approaches, such as dilute acid and steam/pressurized hot water based methods, seek to achieve this by hydrolyzing a significant amount of the hemicellulose fraction of biomass and recovering the resulting soluble monomeric and/or oligomeric sugars. Other pretreatment processes, such as alkaline-based methods, are generally more effective at solubilizing a greater fraction of lignin while leaving behind much of the hemicellulose in an insoluble, polymeric form. Most pretreatment approaches do not hydrolyze significant amounts of the cellulose fraction of biomass, but enable more efficient enzymatic hydrolysis of the cellulose by removal of the surrounding hemicellulose and/or lignin along with modification of the cellulose microfibril structure.

Numerous pretreatment approaches have been investigated at many laboratories, universities, and industrial locations over the past 25 years. In the past, it has been difficult to compare the performance and economics of these various approaches due to difference in feedstocks tested, chemical analysis methods, and data reporting methodologies. Recently, a group of pretreatment researchers across North America have begun to collaborate to investigate different pretreatment approaches on a common basis to allow meaningful comparison. These researchers have formed the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) to advance the efficacy and knowledge base of pretreatment technologies. Current participating institutions in the Biomass Refining CAFI are Auburn University (Y.Y. Lee), University of British Columbia (Jack Saddler), Dartmouth College (Charles Wyman), University of Hawaii (Michael Antal), Michigan State University (Bruce Dale), National Renewable Energy Laboratory [NREL] (Rick Elander), Purdue University (Michael Ladisch), University of Sherbrooke (Esteban Chornet), and Texas A&M University (Mark Holtzapple).

As an active member of the Biomass Refining CAFI, NREL is generating and supplying data on dilute acid pretreatment and the hot wash process to on-going CAFI projects via its ongoing US DOE Biomass Program projects. In addition, NREL is providing several support activities to CAFI projects, including common feedstock supply and analysis, common enzyme supply and analysis, process engineering model development, and training to students of the CAFI institutions. NREL is using pretreatment process information being generated by the Biomass Refining CAFI and other pretreatment researchers to evaluate and ultimately select the most attractive pretreatment approaches for both near term technology platforms (i.e. Enzyme Sugar Platform Project) and for longer term advanced technology platforms. Such evaluation and selection activities are typically made in collaboration with recognized experts in an open, review meeting setting.

Dilute Acid Pretreatment
Biomass Program researchers have been actively developing and demonstrating various dilute acid pretreatment approaches, primarily utilizing sulfuric acid, for more than 15 years. Current work in this area is primarily in support of the Enzyme Sugar Platform Project involving the use of pilot scale pretreatment reactors in the Process Development Unit. Technical goals in this effort are to improve the enzymatic digestibility of cellulose that remains in the pretreated solids by achieving near-complete solubilization of the hemicellulose fraction with resulting high yields of soluble sugars from the hemicellulose. Process economics dictate that such processes be operated at high solids loading to reduce steam demands and produce high concentration streams for subsequent bioconversion and recovery steps of the process.

Hot Wash Process
A variation of dilute acid pretreatment, which may be applicable to other pretreatment processes, involves a high temperature separation and washing of the pretreated solids. It is believed that such a processing step prevents re-precipitation of lignin and/or xylan that may have been solubilized under pretreatment conditions back upon the pretreated solids. This re-precipitation can negatively affect the subsequent enzymatic hydrolysis of the pretreated solids due to the presence of this lignin.

Initial work on this process concept employed a continuously flowing percolation reactor system using extremely dilute sulfuric acid (~0.1% w/v). This process resulted in high yields of recovered xylose oligomers and monomers (>95% of theoretical) and produced significantly more reactive pretreated solids in subsequent enzymatic hydrolysis operations using a hardwood feedstock. In order to minimize required liquid volumes, a hybrid process that employs a batch or high-solids plug-flow pretreatment followed directly by a hot separation and washing of the pretreated solids is being investigated. Pilot scale equipment to operate such a process at the pilot scale is currently being installed in the Process Development Unit.

For Further Reading

Dilute Acid Pretreatment
Schell, D.J., Farmer, J., Newman, N, Dowe, N., McMillan, J.D. (2002). "Dilute Sulfuric Acid Pretreatment of Corn Stover in a Pilot-Scale Reactor: Influence of Compositional Variability." Presented at AIChE 2002 Annual Meeting, Indianapolis, IN, November 3-8, 2002.
Schell, D.J., Farmer, J., Newman, N., McMillan, J.D. (2002). "Dilute Sulfuric Acid Pretreatment of Corn Stover in a Pilot-Scale Reactor: Investigation of Yields, Kinetics, and Solids Enzymatic Digestibilities." (PDF 296 KB) Presented at the 24th Symposium on Biotechnology for Fuels and Chemicals, Gatlinburg, TN, April 28-May 1, 2002.
Tucker, M.P., Farmer, J.D., Keller, F.A., Schell, D.J. (1998). "Comparison of Yellow Poplar Pretreatment Between NREL Digester and Sunds Hydrolyzer." Applied Biochemistry and Biotechnology. 70-72, 25-35.

Hot Wash Process
Washing Away Bioprocessing Cost (Pressurized Hot Wash fact sheet) (PDF 739 KB)
Nagle, N., Elander, R., Newman, M., Rohrback, B., Ruiz, R., Torget, R. (2002). "Efficacy of a Hot Wash Process for Pretreated Yellow Poplar to Enhance Bioethanol Production." Biotechnology Progress. 18, 734-738.
Nagle, N., Torget, R., Newman, M., Rohrback, B., Ruiz, R., Elander, R. (2002). "Aqueous Hot Water Extraction for Enhanced Fractionation of Pretreated Biomass." Presented at AIChE 2002 Annual Meeting, Indianapolis, IN, November 3-8, 2002.
Elander, R., Nagle, N., Torget, R. (2001). "Examining the Potential for Solid Waste and Energy Reduction in Thermochemical Dilute Sulfuric Acid Processes for Biobased Products and Bioethanol Production." Presented at AIChE 2001 Annual Meeting, Reno, NV, November 4-9, 2001.
Torget, R., (2001). "Aqueous Fractionation of Biomass Based on Novel Carbohydrate Hydrolysis Kinetics." U.S. Patent 6,228,177.
Torget, R., Hatzis, C., Hayward, T.K., Hsu, T., Philippidis, G.P. (1996). "Optimization of Reverse-Flow, Two-Temperature, Dilute-Acid Pretreatment to Enhance Biomass Conversion to Ethanol." Applied Biochemistry and Biotechnology. 57-58, 85-101

5:33 上午 | |

 

Selective Enzyme-Mediated Extraction of Capsaicinoids and Carotenoids from Chili Guajillo Puya (Capsicum annuum L.) Using Ethanol as Solvent

J. Agric. Food Chem., 48 (7), 3063 -3067, 2000. 10.1021/jf991242p S0021-8561(99)01242-X Web Release Date: June 17, 2000 Copyright © 2000 American Chemical Society

Selective Enzyme-Mediated Extraction of Capsaicinoids and Carotenoids from Chili Guajillo Puya (Capsicum annuum L.) Using Ethanol as Solvent

R. I. Santamaría, M. D. Reyes-Duarte, E. Bárzana, D. Fernando, F. M. Gama, M. Mota, and A. López-Munguía*
Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62271, Mexico; Facultad de Química, UNAM, Cd. Universitaria, Mexico 04510 D.F., Mexico; and Centro de Engenharia Biológica, IBQF, Universidade do Minho, Largo do Paço, 4709 Braga Codex, Portugal
Received for review November 17, 1999. Revised manuscript received April 14, 2000. Accepted April 17, 2000. This work has been supported by the EC funding INCO-DC (OLNOCO), ERBIC 18CT970206.

Abstract:
The selective extraction of capsaicinoids and carotenoids from chili guajillo "puya" flour was studied. When ethanol was used as solvent, 80% of capsaicinoids and 73% of carotenoids were extracted, representing an interesting alternative for the substitution of hexane in industrial processes. Additionally, when the flour was pretreated with enzymes that break the cell wall and then dried, extraction in ethanol increased to 11 and 7% for carotenoid and capsaicinoid, respectively. A selective two-stage extraction process after the treatment with enzymes is proposed. The first step uses 30% (v/v) ethanol and releases up to 60% of the initial capsaicinoids, and the second extraction step with industrial ethanol permits the recovery of 83% of carotenoids present in the flour.
Keywords: Capsicum annuum; carotenoids; capsaicinoids; selective extraction; enzymatic extraction

Introduction
There is a worldwide growing interest in natural colorants for the food and pharmaceutical industries. As a result, the consumption of synthetic products has declined due to consumer's concern for food safety and stringent regulations from organizations such as the U.S. Food and Drug Administration and the World Health Organization. Carotenoids are one of the most important groups of natural colorants in the food industry, and the genus Capsicum is a widely used source. Capsaicinoids and carotenoids are found in the oily fraction of several varieties of chili peppers (Capsicum annuum). These colorants are normally commercialized as a dried powder such as paprika or as main components of the oleoresin obtained after extraction with hexane. Color and pungency are dependent on capsicum variety. The Guajillo variety is the most frequently used in the Mexican colorant industries due to its low capsaicin content. On the other hand, capsaicin has interesting properties as a therapeutic agent, particularly as a chemoprotector against mutagenesis or tumorigenesis (Surh et al., 1995), and it is also the active ingredient of pepper spray, a relatively safe weapon (Busker and van Helden, 1998).
It has been reported that after hexane extraction, it is possible to reduce pungency in oleoresin by fractionation with 70% v/v methanol solutions, recovering 87% of carotenoids and 83% of capsaicinoids after separation (Balakrishnan and Verghese, 1997). Amaya Guerra et al. (1997) reported the extraction of oleoresin from dried Guajillo pepper with four solvents: ethanol, acetone, ethyl acetate, and hexane. These authors found that the amount of pigment extracted increases with the polarity of the solvent. Hence, ethanol extracted the highest amount of oleoresin, with 82% of capsaicinoids measured in terms of pungency units and 74% of carotenoids. They also reported the fractionation of acetone-extracted oleoresins with a mixture of ethanol/water (9:1) yielding a precipitate with a large proportion of colorants and a solution rich in pungency.

Capsaicinoids are soluble in various solvents such as chloroform, acetone, ethyl ether, ethyl acetate, methyl chloride, ethanol, and 2-propanol, among others (Suzuki and Iwai 1984). Except for the alcohols, these solvents present environmental risks. Nevertheless, due to chemical and toxicological limitations, there is a clear trend in agroindustries to substitute hexane or any other organic solvent for alternative nontoxic solvents, particularly in products for human consumption, with the first option being water or alcohols (Sineiro et al., 1998). In the same context, enzymatic processes have also been proposed to increase yield and selectivity (Domínguez et al., 1994). Ethanol and 2-propanol have been reported in oil extraction from soybean (Baker and Sullivan, 1983), cottonseed (Abraham et al., 1988, 1991, 1993), and sunflower seeds (Sineiro et al., 1998). Water processes have also been developed for the extraction of oil from seeds and vegetables using enzymes to increase the extraction yield (Fullbrook, 1983; Buenrostro and López-Munguía, 1986; Cintra MacGlone et al., 1986; Domínguez et al., 1993, 1994; Shabtai et al., 1998; Rosenthal et al., 1996; Shankar et al., 1997). However, these processes have been applied only to raw materials with oil contents >20%. In some cases dramatic enhancement of oil extraction is obtained with the use of enzymes (Cintra MacGlone et al., 1986). Microscopy has helped to elucidate changes in different plant structures such as oil bodies (Zamski et al., 1987; Napier et al., 1996) and could therefore be applied to analyze the effect of enzymatic action in this kind of process.

In this paper, the extraction conditions of capsaicinoids and carotenoids from Guajillo peppers in ethanol solutions are established. The effect of an enzymatic pretreatment on extraction yield as well as on cell structure by analysis with light microscopy is also established and, finally, a selective extraction for both products in a two-step process is proposed.

Materials and Methods
Quantitative Analysis. Chili Guajillo puya (Capsicum annuum L.) was purchased in a local market in Morelos, Mexico. Samples were also provided by Laboratorios Bioquimex S.A. de C.V. (Mexico). This variety was chosen due to its high carotenoid content. After sun-drying, it was milled and sieved to obtain a flour with a particle size 1.4 mm. Capsaicinoids were determined according to the method of Hoffman (1983) in an HPLC system equipped with a UV detector set at 280 nm and a Bondapack C18 column (3.9 × 300 mm). Acetonitrile/water, 1% acetic acid, 40:60 (v/v), was used as eluent at a flow rate of 1.5 mL/min. Samples were purified in Sep-Pak cartridges according to the method of Attuquayefio and Buckle (1987). Carotenoids were measured in a Beckman spectrophotometer at 450 nm, using = 2500 for quantification after extraction either with ethanol or with ethanol/water mixtures. Under these assay conditions carotenoids are the main product detected (Mínguez-Mosquera et al., 1981). Capsaicin of 90% purity from Sigma was used as standard. Proximate analysis of flour samples resulted in the following composition: humidity, 8.6; protein, 16.5; ashes, 6.5; fat, 8.1; crude fiber, 29.3; and carbohydrates, 31 g per 100 g of flour. Total capsaicinoids were 0.29 and total carotenoids 0.125 g per 100 g of flour and are included in the fat percentage.

Extraction Process. Several extraction options were analyzed in the course of this study. They are generalized in Figure 1. In general, three options were considered: water, industrial ethanol, and ethanol solutions. Water is a poor solvent for these products, but it is required for the enzymatic treatments prior to extraction.

Figure 1 Extraction diagram. Three extraction options were studied: water, industrial ethanol, and ethanol solutions (30% v/v). Extraction yields are reported for each step.
Aqueous Enzymatic Treatment. Various enzymes were selected to soften the tissues in Capsicum peppers either before or during the extraction. All enzymes were from Novo Nordisk Industry: Olivex (an enzyme preparation from Aspergillus aculeatus mainly with pectolytic activity but containing also hemicellulolytic and cellulolytic side activities; 26000 pG/mL), Celluclast (a preparation from Trichoderma reesei with cellulolytic activity; 1500 NCU/g), Viscozyme L (an enzyme preparation from Aspergillus with a wide variety of carbohydrase activities bearing 120 FBG/mL), and Peczyme 5XAL (an enzyme preparation from Aspergillus niger with pectin esterase and arabanase activities, bearing 75000 AJDU/mL and 90000 UA/mL, respectively). Enzymatic action was followed by measuring the release of reducing sugars with the DNS method (Miller, 1959). Treatment with enzyme in water was carried out with dried flour and 1-5%/mL/100 g of flour) of the enzymatic commercial product for 5 h at 50 C, pH 4.5, 120 rpm, and a 1:50 (w/v) flour/water ratio. The paste obtained was dried in a vacuum drier (Precision, Chicago, IL) at 50 C until water was reduced to ~8% humidity. In all cases controls were performed in the absence of enzymes. All of the experiments were performed with three to five repetitions in batch process.

Ethanol Extraction. Extractions were carried out with industrial ethanol (96% purity from ADYDSA, Mexico) both directly from dried flour and from the paste resulting after the aqueous enzymatic treatment. All of the experiments were performed by duplicates and in some cases by triplicates for 7 h at 50 C and a 1:50 (w/v) solid/solvent ratio, in a rotatory shaker (Gyratory Water Bath Shaker model G76) at 120 rpm.

Selective Extraction of Capsaicinoids and Colorants. To selectively extract capsaicinoids and carotenoids, the process was divided into two steps. In the first step, capsaicinoids were extracted from 1:50 (w/v) flour/solvent suspensions with 15 or 30% (v/v) ethanol solutions at 50 C, in a rotatory shaker at 120 rpm; after 7 h, the mixture was filtered. The second batch extraction for the solids obtained was carried out under the same conditions but using industrial ethanol (96%) as solvent to solubilize the largest portion of carotenoids.

Analysis with Light Microscopy. Sample Preparation. Dried chili flour was fixed for 3 h, at 4 C, with a mixture of 2% formaldehyde and 2.5% glutaraldehyde in phosphate buffer (pH 7.0, 0.05 M); 2 mM MgCl2 was added to minimize the loss of lipids from the cells. Fixation was followed by washing with several batches of buffer, also for 3 h. The flour was then dehydrated at 4 C (each step lasting 1 min also to minimize lipid extraction) according to the following procedure: twice with 50% ethanol, a mixture of tert-butyl alcohol/ethanol/water (1:4:5, 2:5:3, 7:10:3, 10:9:0), then with tert-butyl alcohol/absolute ethanol (3:1), and finally twice with tert-butyl alcohol. The dehydrated flour was embedded in tert-butyl alcohol and paraffin (1:1), for 2 h, at 60 C and then twice on paraffin at 60 C, for 1 h. Finally, it was included in paraffin, and sections of 4-6 m were obtained with a microtome model Jung RM2035, from Leica.

Figure 2 Production of soluble reducing sugars after aqueous enzymatic treatment of chili Guajillo puya flour: Viscozyme L (), Olivex (), Celluclast (), and control without enzyme (). Reaction conditions: 50 C, 5 h, pH 4.5, 120 rpm, flour to water relation of 1:50, and 5% v/w enzyme.

Figure 3 Light micrographs of chili flour (magnification 630×; each panel is reproduced at 50% of its original size): (A) nontreated, stained with PAS-Amido Black (proteins stain blue, cellulose stains red); (B) nontreated, stained with Sudan Black (lipids, cutin, suberin, and waxes stain black); (C) nontreated, stained with Saphranin-Picro Aniline Blue (lignin stains red; cellulose stains blue); (D) pretreatment with Viscozyme L; (white arrow) protein bodies; (black arrows) lipid bodies.

Staining. Paraffin was removed from the sections by placing them in xylan (two steps, 2 min each) and then in a mixture of xylan and absolute ethanol (1:1), also for 2 min. Afterward, sections were rehydrated in a graded ethanol series, starting from absolute ethanol to 50% ethanol, 1 min each step (ethanol concentrations decreased by 10% in each step).

Sections were examined with the following methods: Safranin and Picro Aniline Blue, periodic acid Shiff reagent (PAS), Amido Black (Clark, 1981), Coomassie Brilliant Blue R250 (Gahan, 1984), Sudan Black (Bancroft and Stevens 1996; section were stained for 30 min; the saturated staining solutions were also made in 70% propanol). For some of the stained sections, the original mounting media referred to in Clark (1981) was Canada balsam. In this work, Entellan was used to prevent color fading. Pertinent results were the ones obtained with PAS-Amido Black and Sudan and, therefore, are the only ones presented.

Results and Discussion
The feasibility of Capsicum extraction processes has been already demonstrated (Amaya Guerra et al., 1997). We found that 80% of capsaicinoids and 73% of carotenoids are extracted when industrial ethanol (96%) is used as solvent under the experimental conditions described under Materials and Methods, values very similar to those already reported.

Capsaicin has a low solubility in water (Suzuki and Iwai, 1984), so it is not surprising that during the treatment with water at 50 C only 15% of capsaicinoids were found in solution. It has also been observed that some colorants, other than carotenoids, are extracted with water. Mínguez-Mosquera et al. (1992) reported that coloration in Capsicum aqueous extracts is due to a small amount of water-soluble colorants, presumably polyphenols, as the carotenoid fraction is soluble only in organic solvents.

Oil extraction by aqueous processes has been shown to be feasible when enzymes are used to break down vegetable tissue, allowing oil to be released from cells and collected after centrifugation (Buenrostro and López-Munguía, 1986; Cintra McGlone et al., 1986; Domínguez et al., 1994; Rosenthal et al., 1996). For the case of chili flour, various enzymes were active toward some components during the enzymatic treatment, as shown by a reduction in total mass and the increase in soluble reducing power during the first hour of reaction (Figure 2). However, an extraction yield of only 15 ± 2% of capsaicinoids is obtained with or without the addition of different commercial enzymes such as Olivex, Viscozyme L, Peczyme 5XAL, or Celluclast at concentrations as high as 5% (v/w) at 50 C. Even when the temperature was increased to 80 C after the enzymatic treatment, the yield remained low (17 ± 3%). These observations will be further discussed below.

The enzymatic treatments resulted in relatively low cell wall degradation (Figure 3); histological studies were carried out with the chili flour to analyze the cell wall structure/composition and the lipid distribution. Examination of the chili flour without treatment by light microscopy showed that the protein globules detected with Amido Black (Figure 3A) are also stained with Sudan Black (Figure 3B), a specific stain for lipids, cutin, and waxes. Taking into account that the oil content of Capsicum is <8%, href="http://www.geocities.com/isubscribe/journals/jafcau/48/i07/figures/jf991242pf00004.html">
Figure 4 Ethanol extraction of carotenoids from chili guajillo puya flour directly from flour (black bar), after treatment with Viscozyme L 1% (vertically striped bar) and 5% (dotted bar) or Peczyme 5XAL 1% (slashed bar) and 5% (windowpane bar), and after treatment with water (white bar). Reaction conditions: 50 C, 7 h, agitation in a rotatory shaker at 120 rpm, solid to solvent relation of 1:50.

To recover the maximum yield of capsaicinoids and carotenoids in separate fractions, we explored a selective extraction process. Although the separation of both compounds has already been reported by Amaya Guerra et al. (1997) and Balakrishnan and Verghese (1997), the proposed process requires the extraction of the oleoresin. As shown in Figure 5, when water is substituted with up to 30% (v/v) ethanol solutions, 62 ± 0.1% of capsaicinoids may be extracted in the first step with a low removal of carotenoids (1.4%). Afterward, in a second extraction with industrial ethanol, 70% of carotenoids and 26% of capsaicinoids are recovered. If an enzymatic pretreatment with Viscozyme L is included in the process, then the final carotenoid extraction yield increases to 83 ± 1%. This is equivalent to an increase in the carotenoid extraction in the second step of 10 ± 1%.

Figure 5 Two-step process for the selective extraction of capsaicinoids and carotenoids. In the first step, capsaicinoids are extracted with water (black bar), 30% (v/v) ethanol (white bar), and 30% v/v ethanol after treatment with Viscozyme 5% (slashed bar). (A) In the second step carotenoids in all meals from the first step are extracted with 96% ethanol (B). Reaction conditions: 50 C, 7 h, agitation at 120 rpm, and a solid to solvent relation of 1:50.

These results demonstrate that ethanol is an interesting alternative to hexane for the extraction of valuable products from Capsicum, as it may be used directly for the extraction of 80% of capsaicinoids and 73% of carotenoids with a higher carotenoid extraction yield if the flour is previously treated with enzymes in an aqueous process. However, the best alternative is the selective extraction of both components in a three-step process: (I) pretreatment of the flour with an enzyme solution of Viscozyme L at 5%; (II) a first extraction stage with 30% v/v ethanol to yield 60% of capsaicinoids (from both phases, water and 30% ethanol); and (III) a second extraction stage with ethanol, during which most of the carotenoids (83%) are obtained.
Despite the small increase in the extraction of carotenoids (~10%), a two-step treatment coupled with enzyme pretreatment may represent a significant economical alternative when its application to industrial processes is considered.

Acknowledgment
We are grateful to Novo Industry A/S for the enzymes and to Laboratorios Bioquimex for the supply of samples and information.
* Author to whom correspondence should be addressed [telephone (52)(73) 116265; fax (52)(73) 172388; e-mail agustin@ibt.unam.mx].
Instituto de Biotechnolgía.
Facultad de Química.
Centro de Engenharia Biológica.

Abraham, G.; Hron, R. J., Sr.; Koltun, S. P. Modeling the solvent extraction of oilseeds. J. Am. Oil Chem. Soc. 1988, 65, 129-135.
Abraham, G.; Decossas, K. M.; Hron, R. J., Sr.; Kuk, M. S. Process engineering economic evaluation of the ethanol extraction of cottonseed: preliminary analysis. J. Am. Oil Chem. Soc. 1991, 68, 418-421.[CAS]
Abraham, G.; Hron, R. J., Sr.; Kuk, M. S.; Wan, P. J. Water accumulation in the alcohol extraction of cottonseed. J. Am. Oil Chem. Soc. 1993, 70, 207-208.[CAS]
Amaya Guerra, C. A.; Serna Saldívar, S. R. O.; Cárdenas, E.; Nevera Muñoz, A. Evaluation of different solvent systems for the extraction and fractionation of oleoresins from guajillo peppers. Arch. Latinoam. Nutr. 1997, 47, 127-130.[CAS][Medline]
Attuquayefio, V. K.; Buckle, K. A. Rapid sample preparation method for HPLC analysis of capsaicinoids in capsicum fruits and oleoresins. J. Agric. Food Chem. 1987, 35, 777-779.[CAS]
Baker, E. C.; Sullivan, D. A. Development of a pilot-plant process for the extraction of soy flakes with aqueous isopropyl alcohol. J. Am. Oil Chem. Soc. 1983, 60, 1271-1277.[CAS]
Balakrishnan, K. V.; Verghese, J. Studies on the recovery of pungency-free colour matter from Indian capsicum extracts. Acta Alimentar. 1997, 26, 9-21.
Bancroft, J. D.; Stevens, A. Theory and Practice of Histological Techniques; Churchill Livingstone: New York, 1996; p 220.
Buenrostro, M.; López-Munguía, C. A. Enzymatic extraction of avocado oil. Biotechnol. Lett. 1986, 8, 505-506.[CAS]
Busker, R. W.; van Helden, H. P. Toxicologic evaluation of pepper spray as a possible weapon for the Dutch police force: risk assessment and efficacy. Am. J. Forensic Med. Pathol. 1998, 19, 309-316.[Medline]
Cintra MacGlone, O.; López-Munguía, C. A.; Vernon Carter, J. Coconut oil extraction by a new enzymatic process. J. Food Sci. 1986, 51, 695-697.[CAS]
Clark, G. Staining Procedures, 4th ed.; Williams & Wilkins: Baltimore, MD, 1981; pp 317, 323, 325-327.
Domínguez, H.; Núñez, M. J.; Lema, J. M. Oil extractability from enzymatically treated soybean and sunflower: range of operational variables. Food Chem. 1993, 46, 277-284.[CAS]
Domínguez, H.; Núñez, M. J.; Lema, J. M. Enzymatic pretreatment to enhance oil extraction from fruits and oilseeds: a review. Food Chem. 1994, 49, 271-286.[CAS]
Fullbrook, P. D. The use of enzymes in the processing of oilseeds. J. Am. Oil Chem. Soc. 1983, 60, 476-478.
Gahan, P. B. Plant Histochemistry and Cytochemistry, An Introduction, 1st ed.; Academic Press: London, U.K., 1984; pp 190, 204.
Hoffman, P. G.; Lego, M. C.; Galetto W. G. Separation and quantification of red pepper major heat principles by reverse-phase high-pressure liquid chromatography. J. Agric. Food Chem. 1983, 31, 1326-1330.[CAS]
Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426-428.
Mínguez-Mosquera, M. I.; Fernández-Díez, M. J. Pimiento rojo Español maduro (genero Capsicum). Relación entre los pigmentos carotenoides rojos y amarillos. Grasas Aceites 1981, 32 (5), 293-298.
Mínguez-Mosquera, M. I.; Jarén-Galán, M.; Garrido-Fernández, J. Color quality in paprika. J. Agric. Food Chem. 1992, 40, 2384-2388.[CAS]
Napier, J. A.; Stobart, A. K.; Shewry, P. R. The structure and biogenesis of plant oil bodies: the role of the ER membrane and oleosin class of proteins. Plant Mol. Biol. 1996, 31, 945-956.[CAS][Medline]
Rosenthal, A.; Pyle, D. L.; Niranjan, K. Aqueous and enzymatic processed for edible oil extraction. Enzymol. Microb. Technol. 1996, 19, 402-420.
Shabtai, Y.; Gotshal, N.; Ramot, O.; Wisniak, J. Enzymatic pretreatment of jojoba seeds to facilitate oil extraction. Indian J. Chem. Technol. 1998, 5, 124-130.[CAS]
Shankar, D.; Agrawal, Y. C.; Sarkar, B. C.; Singh, B. P. N. Enzymatic hydrolysis in conjunction with conventional pretreatments to soybean for enhanced oil availability and recovery. J. Am. Oil Chem. Soc. 1997, 74, 1543-1547.[CAS]
Sineiro, J.; Domínguez, H.; Núñez, M. J.; Lema, J. M. Ethanolic extraction of sunflower oil in a pulsing extractor. J. Am. Oil Chem. Soc. 1998, 75, 753-754.[CAS]
Surh, Y. J.; Lee, R. C. J.; Park, K. K.; Mayne, S. T.; Liem, A.; Miller, J. A. Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodimethylamine. Carcinogenesis 1995, 16, 2467-2471.[CAS][Medline]
Suzuki, T.; Iwai, K. Alkaloids; Academic Press: New York, 1984; Vol. 23, pp 227-299.
Zamski, E.; Shoham, O.; Palevitch, D.; Levy, A. Ultrastructure of capsaicinoid-secreting cells in pungent and nonpungent red pepper (Capsicum annuum L.) cultivars. Bot. Gaz. 1987, 148, 1-6.

5:22 上午 | |