in potato and sweetpotato Nutrition and Quality Laboratory International Potato Center (CIP) I. Potato 1. Sampling and sample preparation 1.1. Minerals and phenolic compounds 1.1.1. Sampling Collect 7-10 tubers at random per field replication for each genotype. Each sample comes from each replication. 1.1.2. Sample preparation (adapted from Norbotten, 2000) Wash the tubers with abundant tap water (trying to remove any soil residue), rinse deionazed and distilled water and dry the tubers with towel paper. Peel the tubers and cut each longitudinally in 4 sections With two opposite sections of each tuber prepare the sample for mineral analysis. With the help of a stainless steel slicer, obtain 2 3 slices of each section until obtaining a 50 g weighed sample (take note of the exact fresh weight), place the sample in a glass petri dish and oven dry at 80 ºC. Weight the dried sample, mill in an stainless steal mill, place in kraft paper bags and send to Adelaide University, Australia. With the other 2 opposite sections of each tuber prepare the sample for polyphenolic and chlorogenic acid analysis. Obtain 3 4 slices of each section until having a 100 g weighed sample (take note of the exact fresh weight). Place the sample in polyethylene bags, store at 20ºC and freeze dry. Weigh the dried sample, mill in a stainless steal Thomas mini mill (40 mesh) and store at 20ºC until the analysis. 1.2. Vitamin C 1.2.1. Sampling Collect 5 tubers at random per field replication for each genotype. Each sample comes from each replication. 1.2.2. Sample preparation (adapted from Norbotten, 2000) Wash the tubers with tap water and rinse with still or deionazed water.
1.3. Carotenoides Procedures for nutrient analysis Working paper 2 Cut the tubers longitudinally into 4 sections. Take two opposite sections of each tuber and with the help of a slicer obtains 3 or 5 slices of each section. Mix the slices and take 10g for determining the matter content and 15 g to analyze the vitamin C immediately. 1.3.1. Sampling Collect 5 tubers at random per field replication for each genotype. Each sample comes from each replication. 1.3.2. Sample preparation Wash the tubers with tap water and rinse with still or deionazed water. Peel the tubers, cut in small squares and place then on a blender and homogenize during 2 min approximately. 2. Nutrient analysis 2.1. Minerals Analytical sub-samples of 0.6 g of each samples are taken and digested at 140 C in 70% (w/w) HNO 3 /HClO 4. The mineral concentrations were determined by inductively coupled plasma optical emission spectrophotometry (ICP-OES) using an ARL 3580B ICP. As Al is commonly found in higher levels in the soil and in very small levels in crops, it is also analyzed to give an indication of Fe contamination from soil or dust particles. Note: This step is done in Wayte Analytical Services, Adelaide University, Australia. 2.2. Vitamin C Adapted from Egoaville et al., 1988. Reagents preparation Extracting solution: Oxalic acid 0.4%, acetone 20% Dissolve 4 g of oxalic acid in 100 ml of still water (agitate with the help of a magnet bar until dissolve completely). Add still water until 500 ml and add 200 ml of acetone. Agitate slowly and carry to ph 1.1 with concentrated sulfuric acid (to avoid acetone evaporation cover the beaker with aluminum foil). Finally transfer the extracting solution to a volumetric flask and carry to 1 liter with still water.
Working paper 3 Stock solution of 2,6 dichloroindophenol (DCIP) Dissolve 100 mg of 2,6 DCIP in 100 ml of warm water (40-50ºC). Add 84 mg of NaHCO 3, shake with the help of a magnetic bar, transfer the solution to a volumetric flask and carry to 500 ml with still water. Filter with suction and store the solution in an amber flask. All this preparation should be done avoiding the light. For its using, the stock solution should be diluted with still water such that when 1 ml of the extracting solution and 9 ml of DCIP solution are reacted, an absorbance value (at 520 nm) of 0.300 to 0.350 is obtained. This range of absorbance is usually obtained diluting 1 ml of the stock solution of 2,6 DCIP in 13 ml of still water. Stock solution of ascorbic acid Dissolve 100 mg of ascorbic acid in 50 ml of extracting solution. Carry to 100 ml using an amber volumetric flask. Preparation of the ascorbic acid standard curve Prepare ascorbic acid standard solutions of ascorbic acid with concentrations of 10, 20, 30, 40 and 50 mg/ml. Take aliquots of 1, 2, 3, 4 y 5 ml of the stock solution and carry to 100 ml with the extracting solution. Analyze 1 ml of each one of the prepared standards as follow: Analysis Mix 1 ml of the extracting solution with 9 ml of the diluted dye (2,6 DCPI) and read the absorbance (Reagent blank) at 520 nm (read 3 times) Mix 1 ml of the standard with 9 ml of the diluted dye (2,6 DCPI) and after 1 minute read the absorbance (Standard absorbance) at 520 nm (read 3 times). Mix 1 ml of the standard with 9 ml of still water and read the absorbance at 520 nm (Standard blank). Subtract the sample blank from the sample absorbance and then subtract this value from the reagent blank. This final value (Real absorbance) is used to build the standard curve (Real absorbance vs concentration). Weigh 15g of the homogenized fresh sample in the extracting glass, add 75 ml of the extracting solution and mix in the Sorvall Omni Mixer during 5 minutes. Rinse the mixer with the extracting solution and filter the extract through filter paper Whatman # 2 using the vacuum pump. Transfer the filtered extract into an amber volumetric flask, carry to 100 ml and mix. Mix 1 ml of the extracting solution with 9 ml of the diluted dye (2,6 DCPI) and read the absorbance (Reagent blank) at 520 nm (read 3 times) Mix 1 ml of the extract with 9 ml of the diluted dye (2,6 DCPI) and after 1 minute read the absorbance (Sample absorbance) at 520 nm (read 3 times). Mix 1 ml of the extract with 9 ml of still water and read the absorbance at 520 nm (Sample blank).
Working paper 4 Subtract the sample blank from the sample absorbance and then subtract this value from the reagent blank. This final value (Real absorbance) is used to estimate the concentration of vitamin C using as a reference the ascorbic acid standard curve. 2.3. Phenolic compounds (Adapted from Current Protocols in food analytical chemistry (2002) I1.1.1- I1.1.8) Reagent preparation Extracting solution: Ethanol 80% Place 200 ml of still water in a 1 liter volumetric flask, bring to 1 l with ethanol mix very well and transfer the extracting solution to a flask closing tightly to avoid evaporation. Sodium carbonate solution (20%) Dissolve 50g of sodium carbonate anhydrous in 200 ml of still water and boil the solution. Let the solution gets the environment temperature and add 1 spoon of sodium carbonate for saturate the solution. After 24 hours, filter the solution and bring it to 250 ml. Transfer the solution to an amber glass. Stock solution of chlorogenic and gallic acid Dissolve chlorogenic or gallic acid in 5 ml of ethanol and bring to 50 ml with still water. Standard curve preparation Prepare chlorogenic and gallic acid standard solutions with concentrations of 50, 100, 200, 300, 400 y 500 mg/ l. Take aliquots of.5, 1, 2, 3, 4 y 5 ml of the stock solution and carry to 50 ml with water. Analyze the standards as follow: Analysis Prepare each standard in duplicate and also prepare a blank reagent. Place 100 ul of the standard and 100 ul of water in the case of still water in glass tubes and add 8 ml of still water and 500 ul of the Folin reagent, mix and leave for 6 min. Add 1500 ul of the saturated sodium carbonate solution and mix very well. Place the tubes in a water bath at 40 C during 30 min. Read at 765 nm. Weigh 1 g of milled freeze-dried sample and place in a glass. Add 15 ml of extracting solution, stir during 20 min with hot temperature until the sample boils. Filter the extracted solution in a volumetric flask of 25 ml using filter paper (Whatman #2).
Working paper 5 Add 10 ml of extracting solution and re-extract the sediment by stir during 20min under hot temperature. Filter the re-extracted solution and rinse the glass with the extracting solution and filter again. Bring to 25 ml with still water. Prepare each sample in duplicate and also prepare a blank reagent (BR) Place 100 ul of the sample and 100 ul of water in the case of still water in glass tubes and add 8 ml of still water and 500 ul of the Folin reagent, mix and leave for 6 min. Add 1500 ul of the saturated sodium carbonate solution and mix very well. Place the tubes in a water bath at 40 C during 30 min. Read at 765 nm. The end concentration of phenolic compounds is estimated based on the chlorogenic and gallic acid standard curves. 2.4. Carotenoids (Adapted from the maize protocol of Rodriguez-Amaya and Kimura, 2004) Carotenoid extraction Weigh a portion (6-8 g for the more pale yellow samples and 2-5 g for the more intense yellow samples) of the homogeneous, representative sample in a beaker. The weight depends on the intensity of the color of the sample. Add 30 ml approximately of cold acetone, homogenize in a Turrax during 1 min at 21500 rpm and filter with suction through a sintered glass funnel, receiving the extract in a protected suction flask. Wash the turrax and residue with small amounts of acetone. Repeat the extraction until the residue is devoid of color. Usually only 3 extractions are required. Partition to petroleum ether Put petroleum ether (30 ml approximately) and a small amount of still water in a 500 ml separatory funnel. Transfer the acetone extract in 4 or more steps depending on the color intensity of the extract. After each transference, add 200-250 ml of still water letting it flow allow along the walls of the separatory funnel. Let the 2 phases separate and discard the lower, aqueous phase every time. Wash 3-4 times with 100-150 ml of a 5% sodium chloride solution to remove residual acetone. Let the 2 phases separate during 4 minutes and discard the aqueous phase. The sodium chloride solution is used to avoid the formation of an emulsification. In the last washing let the 2 phases separate 10 minutes and be sure to completely discard the lower phase with discarding any of the upper phase. Saponification Collect the transferred extract in a 125 ml Erlenmeyer, add 0.025g of antioxidant BHT (2,6-Di-tert-butyl-4-methylphenol).
Working paper 6 Add an equivalent volume of 10% methanolic KHO to the extract in the flask and mix. Cover the flask with aluminum foil and let the mixture stand in the dark at room temperature overnight (16-20 h). Transfer of the saponified extract to petroleum ether The next day, transfer the saponified extract to a 500 ml separatory funnel. Collect the methanolic phase in the same flask. Wash the walls of the separatory funnel tip and flask with acetone. Add an equivalent volume of acetone to the collected methanolic phase. Add an equivalent volume of dietilic ether to the ethereous phase. Transfer the methanolic phase in various fractions. The number of transfers depends on the color intensity of the extract. After each transfer, shake the separatory funnel and add 200-250 ml of distilled water letting it flow for the walls. Let it stand 4 minutes. In the last transfer wash the separatory funnel with acetone. Wash 5 times with 100-150 ml of distilled water. Let the 2 phases separate during 4 minutes and discard the aqueous phase. In the last washing let the 2 phases separate 10 minutes and be sure to completely discard the lower phase with discarding any of the upper phase. Filtering Filter the ethereal extract through a glass funnel containing anhydrous sodium sulfate and collect in a 25 ml volumetric flask. If the ethereal extract contains a volume higher than 25 ml dry with N gas. Bring to 25 ml with petroleum ether. Use 15 ml of the extract in a tube for HPLC analysis and use the other 10 of spectrophotometric determinations of total carotenoids at 450 nm. Introduction to the HPLC equipment Place 15 ml of he extract in a tube. Place the tube in warm water (35 ºC) and dry the extract with nitrogen gas (N2). Storage at 20ºC and immediately before injection thaw and redissolve in 1 ml of HPLC grade acetone, filter through a 0.22 um PTFE syringe filter (Millipore) directly into sample vials and inject 10 ul into chromatograph. HPLC chromatographic conditions Polymeric C30 column: YMC C30, 3um, 4.6 x 250 mm Mobile phase: methanol:methyl-tert-butyl ether Gradient elution: 95:5 to 80:20 in 30 min, then to 60:40 in 40 min and finally to 40:60 in 60 min. Flow rate: 0.8 ml/min, reequilibration 15 min.
Working paper 7 II. Sweetpotato 1. Sampling and sample preparation 1.1. Sampling Collect 5 roots (at random) per replication for each genotype. Each sample comes from each replication. 1.2. Sample preparation (adapted from Norbotten, 2000) Wash the roots with abundant tap water (trying to remove any soil residue), rinse deionazed and distilled water and dry the roots with towel paper. Peel the roots and cut each longitudinally in 4 sections. With the help of a stainless steel slicer, obtain 3 4 slices of each of two opposite sections of each root obtaining a 50 g weighed sample (take note of the exact fresh weight). Place the sample in polyethylene bags, store at 20ºC and freeze dry. Weigh the dried sample, mill in an stainless steal mill and store at 20ºC until the analysis. 3. Nutrient analysis 3.1. Chemical analysis 3.1.1. Mineral analysis Analytical sub-samples of 0.6 g of each samples are taken and digested at 140 C in 70% (w/w) HNO 3 /HClO 4. The mineral concentrations were determined by inductively coupled plasma optical emission spectrophotometry (ICP-OES) using an ARL 3580B ICP. As Al is commonly found in higher levels in the soil and in very small levels in crops, it is also analyzed to give an indication of Fe contamination from soil or dust particles. Aluminum levels higher than 6 ppm, in dry weight basis are indicators of soil contamination. Note: This step is done in Wayte Analytical Services, Adelaide University, Australia. 3.1.2. Total carotenoid and -carotene analysis (adapted from Rodriguez- Amaya and Kimura, 2004) Carotenoids extraction Weigh a portion (0.1-0.5g) of the freeze-dried sweetpotato powder (the weight depends on the carotenoid content of the sample).
Working paper 8 Place the weighed sample in a mortal and add 30 ml of cold acetone, homogenize 1 minute using a polytron homogenizer (or a vortex) and filter with suction through a Buchner funnel with filter paper or sintered glass funnel. Wash the flask, polytron homogenizer and residue with acetone, receiving the washing in the funnel and if necessary, repeat the extraction procedure. Partitioning to petroleum ether Put 50 ml of petroleum ether in a separate funnel and add a small portion of the acetone extract. Add distilled water slowly, letting it flow along the walls of the funnel. To avoid formation of an emulsion, do not shake (once formed, an emulsion can be broken by adding acetone or sodium chloride). Let the two phases separate and discard the lower aqueous-acetone phase. Add another portion of the acetone extract and repeat the operation until all of the extract has been transferred to petroleum ether, then wash 4-5 times with water to remove residual acetone. Collect the petroleum ether phase in a 100-ml volumetric flask making the ethereal extract pass through a glass funnel containing a anhydrous sodium sulfate. Total carotenoid determination Read the carotenoid ethereal extract at 450 nm and calculate the total carotenoid content with the following formula: A y (ml) 10 6 x ( g) = A 1% 100 1cm x ( g) x ( g/g) = Weight of sample (g) Where x is the weight or concentration of the carotenoid, y is the volume of the solution that gives an absorbance (A) at 450 nm and A 1% 1cm is the absorption coefficient of - carotene in petroleum ether (2592). HPLC determination Introduction to the HPLC equipment Concentrate the extract in a rotary evaporator (t<35ºc).
Working paper 9 Dry under N2, and immediately before injection, redissolve in 1 ml of HPLC grade acetone, filter through a 0.22 mm PTFE syringe filter (Millipore) directly into samples vials and inject 10 ul into the chromatograph. HPLC chromatographic conditions Monomeric C18 column: Waters Spherisorb ODS 2, 3 um, 4.6 x 1150 mm. Mobile phase: acetonitrile:methanol:ethylacetate (0.05 % triethylamine). Isocratic elution: 80:10:10, flow rate: 0.7 ml/min. Calculation Calculate carotenoid concentration using the formula: C x (ug/g) = A x x Cs (ug/ml) x total volume of extract (ml) A s x sample weight (g) Where C x = concentration of carotenoid X; A x = peak of area of carotenoid X; Cs = concentration of the standard; A s = peak area of the standard. 1.1. NIRS analysis Mineral, total carotenoid, -carotene and protein concentration in sweetpotato are estimated by Near Infrared Reflectance Spectroscopy in a FOSS NIRS Systems 6500 scanning monochromator (400-2500 nm).
Working paper 10 References Egoaville, M. J., J.F. Sullivan, M. F. Kozempel and W. Jones. J. 1988. Ascorbic acid determination in processed potatoes. American Potato Journal. Vol 65:91-97. Rodriguez-Amaya, D. and K. Mieko. 2004. Harvestplus Handbook for carotenoid analysis. HarvestPlus Technical Monograph 2. Washington, DC and Cali: International Food Policy Reasearch Institute (IFPRI) and International Center for Tropical Agriculture (CIAT). Norbotten, A., E.B. Loken and A.H. Rimestad. 2000. Sampling of potatoes to determine representative values for nutrient content in a national food composition table. J. Food. Compos. Anal. 13:369-377. Waterhouse A. 2002. Current Protocols in Food Analytical Chemistry. University of California, Davis, U.S.A. I1.1.1-I.1.1.8
Working paper 11 Annex 1. Avoiding sample contamination Washing your hands with tap water and rising with deionazed and distilled water every time you are going to manipulate the sample. Using stainless steal equipments and material. Cleaning the working tables and material with nitric acid 0.1% and rinsing with deionazed and distilled water. Avoiding people who are not involve with sample preparation get into the sample preparation room (specially people coming from the field). Nitric acid 0.1% preparation: Dilute 1.5 ml nitric acid in 1 liter distilled water Nitric acid 65% x ml = Nitric acid 0.1% x 1000ml ml = 1.53 ml
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