Analytical Methods

2.3.3.1. Monosaccharide and Disaccharide analysis (Gas Chromatography)

2.3.3.1.a. Potato Sample Preparation

As developed for potato samples by Dr Ian Andrews of Massey University.

60-100 mg dried, ground potato sample was weighed into a glass 15 mL centrifuge tube (with screw cap).

5 mL of 80% ethanol was added to the tube, capped and then mixed well on a vortex mixer to suspend the sample uniformly (samples that required initial dissolving had 1 mL water added to the sample first, were mixed thoroughly using a vortex mixer and then had 4 mL absolute ethanol added and then remixed). Samples were heated for 5 min in a waterbath at 80oC to extract soluble sugars, cooled, and then 5 mL 80% ethanol was added to make 10 mL total. Samples were centrifuged at 3000rpm (x 1942 g) for 5 min in a benchtop centrifuge (Heraeus Labofuge 400R, Germany) and the supernatant was used directly for the assay.

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2.3.3.1.b. Preparation of trimethylsilyl (TMS) Sugars

As developed for potato samples by Dr Ian Andrews of Massey University.

0.05 mL of xylitol internal standard (10 mg/mL) and 0.4 mL of sample supernatant prepared above were added sequentially into a 1 mL glass screw-cap vial. At the same time a control (0.45 mL 80% ethanol) and sugar standards (glucose, fructose, galactose, sucrose, maltose and lactose) were prepared (0.1 mL of 10 mg/mL standard added to 0.35 mL 80% ethanol). Each sample, control and standard was placed in a heating block and the ethanol carefully evaporated off under a stream of air or nitrogen to obtain a glassy syrup. 0.05 mL of TMS reagent (1:1, v/v mixture of TMS-imidazole in dry pyridine) was added to each and if necessary, samples were heated at 50o to dissolve sugars. 0.2 mL of Hexane was then carefully added, and the samples mixed using a vortex mixer. The samples were allowed to separate into layers (or where necessary, samples were transferred to a microfuge tube, fitted with autosampler vial inserts and centrifuged. 2μL of the samples prepared above were injected into a Hewlett Packard 5890 Gas Chromatograph fitted with a ZB-1 (Phenomenex, Zebron, 100% polydimethylpolysiloxane) capillary column (30 m x 0.25 mm x 0.5 μm). Samples were run using Nitrogen as the carrier gas (2 mL/min constant flow) and an oven programme of 160o - 300oC at 10o C/min until the temperature reached 300oC.

2.3.3.2. Total Sugar Estimation by Phenol-Sulphuric assay

This method was an adaptation of the methods described by Dubois et al. (1956) and Immers (1964). Samples prepared as in 2.3.3.1.a. above were diluted tenfold with water and 0.4 mL duplicates were added into clean round-bottom glass test tubes, (150 mm x 18 mm internal diameter). 0.4 mL glucose standards (100 μg/mL or 200 μg/mL) and a 0.4 mL water reagent blank were prepared. In a fumehood, 0.4 mL of 5% phenol (aqueous) was added to the prepared tubes and mixed using a vortex mixer. 2.4 mL concentrated sulphuric acid was added quickly and directly into the sample at the bottom of the tube and then the tubes were heated in a boiling waterbath at 100oC for 5 min to ensure uniform reaction. Once cooled, the absorbance of the solution in the tubes was read at 490 nm against the reagent blank.

Where the sample was strongly coloured (such as with Tūtaekuri samples), it was necessary to use controls without phenol and subtract these from the test sample absorbance. Standards were also subjected to the same treatment, such that standards were prepared with and without phenol.

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For the controls without phenol, 0.4 mL water was substituted for the phenol (0.8 mL total volume of water in the control used for measuring against Tūtaekuri). Total sugar was expressed as grams of sugar (glucose) per 100 g of fresh weight potato sample.

2.3.3.3. Fibre Components

Total dietary fibre, insoluble dietary fibre and soluble dietary fibre were determined using the enzymatic-gravimetric method (AOAC 991.43).

2.3.3.4. Amino Acid Analysis

Amino acid (AA) analysis includes an estimation of the amino acids present in whole protein as well as free amino acids. Eighteen amino acids: histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), cysteine (Cys), tyrosine (Tyr), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), valine (Val), alanine (Ala), arginine (Arg), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), proline (Pro) and serine (Ser) were determined in the freeze-dried potato material described earlier (Section 2.2.2.1.). All amino acids except Cys, Met and Trp were analysed by hydrolysis with 6M hydrochloric acid followed by HPLC separation (AOAC 994.12). Cysteine and methionine were analysed using performic acid oxidation, followed by HPLC separation (AOAC 994.12), while Tryptophan was analysed using alkaline hydrolysis followed by HPLC separation (AOAC 988.15).

2.3.3.5. Fatty Acid Analysis

Fatty acid determination by the conversion of non-volatile fatty acid compounds into volatile fatty acid methyl esters (FAMES) was carried out on dried potato samples using the procedure developed by Sukhija and Palmquist (1988).

The fatty acids tested in this research included:

Saturates: all cis: c4:0, c6:0, c8:0, c10:0, c11:0, c12:0, c13:0, c14:0, c16:0, c17:0, c18:0, c20:0, c21:0, c22:0, c23:0, c24:0.

Monounsaturates: all cis: c14:1n5, c15:1n5, c16:1n7, c17:1, c18:1n9, c20:1n11, c22:1n9, c24:1n15 and c18:1-trans elaidic acids.

Di/Polyunsaturates: all cis: c18:2n6, c18:2-trans linoelaidic, c18:3n3, c18:3n6, c20:2n6, c20:3n6, c20:3n3, c20:4n6, c20:5n3, c22:2n6, c22:6n3 acids.

Where there were no, or <1 mg fatty acid per 100 g fresh weight potato material detected in all cultivars, the results were not included in the tables unless specifically mentioned.

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2.3.3.6. Mineral Analysis

Zinc (Zn), copper (Cu) and manganese (Mn) were measured by nitric/hydrochloric acid digestion followed by Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) analysis; selenium was determined by sample digestion with aqueous trimethylammonium hydroxide (TMAH) at 90oC followed by ICP-MS determination. Potassium (K), phosphorus (P), magnesium (Mg), calcium (Ca), sodium (Na) and iron (Fe) were analysed using nitric/hydrochloric acid digestion, followed by Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES) determination. Mineral analysis was carried out on freeze-dried samples (see Section 2.2.2.1. for material preparation) by AgResearch Ltd, Palmerston North, New Zealand.

2.3.3.7. Selected Vitamins

Based on general estimations of vitamin content in potato tubers (Lisinka and Leszcyznski, 1989), the consumption of potatoes in New Zealand (Horticulture NZ, 2006; Ministry of Health, 2011), and thus their potential nutritional value, selected vitamins were chosen to be analysed in the potato tubers in this trial. These included Vitamin C, Vitamin B1 (Thiamine HCl), Vitamin B6 (Pyridoxal Phosphate), Folic Acid and Niacin.

Analysis of all Vitamins was carried out by AsureQuality New Zealand, Auckland, New Zealand. Vitamin C was measured using the liquid chromatography and fluorescence detection method (Dodson et al., 1992); Thiamine by HPLC (European Committee for Standardisation (CEN), 2001); Vitamin B6 by HPLC (Olds et al., 1993; Bitsch and Möller, 1989); Folic Acid by Optical Biosensor Based Immunoassay (Indyk et al., 2000) and Niacin by HPLC (Woollard, 1984).

2.3.3.8. Limits of Uncertainty

Uncertainties of measurements for nutrients tested as part of the analyses described in Section 2.3 are as follows: Insoluble dietary fibre, soluble dietary fibre (±6%); sugars(±5%); histidine (±6.9%); isoleucine (±5.56%); leucine (±4.96%); lysine (±6.23%); methionine (±6.58%); cysteine (±6.8%); tyrosine (±4.54%); phenylalanine (±4.74%); threonine (±10%); tryptophan (±6.52%); valine (±5.73%); alanine (±5.07%); arginine (±5.81%); aspartic acid (±5.18%); glutamic acid (±4.4%); glycine (±5.68%); proline (±5.87%); serine (±6.59%); fatty acids (± 5%); vitamin C (±11.64%); vitamin B1 (±15.42%); vitamin B6 (±16.48%); niacin (±15.42%); folate (±9.77%).

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