The Effect of Storage on the Amino Acids Composition in Potato Tubers

ČERNÁ, M., KRÁČMAR, S.: The eff ect of storage on the amino acids composition in potato tubers. Acta univ. agric. et silvic. Mendel. Brun., 2010, LVIII, No. 5, pp. 49–56 The aim of this study was to investigate the eff ect of storage duration on amino acids content in potato tubers. Tubers of six cultivars were stored for 16 weeks. Crude protein was determined by the Kjeldahl method. The amino acid content was identifi ed a er acid hydrolysis; sulphur amino acids were oxidized with the mixture of hydrogen peroxide, formic acid and phenol. The essential amino acids index and chemical score was calculated. The whole egg protein was used as reference protein. The effect of storage duration and cultivar on the total amino acids content was found to be signifi cant in all potato varieties. The level of the total amino acids contents was determined within the range of 80– 87%. Similar downward trend was observed in crude protein. The essential amino acids index varied considerably among the potato cultivars, ranging from 42 to 57%. Sulfur amino acids and isoleucin were the limiting amino acids in all the investigated tubers. Solanum tuberosum L., storage, amino acid, protein, essential amino acids index Potato (Solanum tuberosum L.) is a typical crop grown in many countries in Europe. In the Czech Republic it is one of the most important horticultural crops. More than 753 thousand tons of potatoes were harvested in 2009 (ČSÚ, 2010). Potatoes are o en perceived as a mere starch source, but they are also rich in minerals, vitamins and protective substances. Potatoes are used in diets, namely for their good diges ti bi li ty and a low allergen potential. Food in to leran ce can be triggered only by potato products like chips and crisps. It is a well known fact that potato crops produce a higher quantity of protein per hectare than cereals (FAO, 2009). Potatoes contain about 2% of crude protein in fresh matter. Because of rela ti ve ly higher potato consumption (annually approx. 70 kg per capita in the Czech Republic), potato tubers are an important source of protein in a human diet. However, the amount of crude protein depends on cultivar, agricultural practices and agroclimatic factors. Crude protein includes a number of nitrogen fraction, amides and free amino acids (Eppendorfer et al., 1979). Potato protein is valuable for a high biological value. The biological value of potatoes mixed with eggs reaches to the amount of about 140% of standard protein (Zrůst, 2004). The basic role of potato protein in nutrition is the contribution to suffi cient amounts of essential amino acids for protein endogenous synthesis by human body. It is essential to take the diff erence between nutritional and biological aspects in consideration. Therefore as far as biological aspect is concerned, nonessential amino acids are more important than the essential ones (Murray et al., 1998). Chemical score obtained by comparing the limiting amino acids in relation to the reference protein is a chemical method which refl ects the protein quality (Friedman, 1996). Total amino acids content mainly depends on cultivar (Zrůst, 2004). Previous studies report variab le trends in free amino acids contents in potato during the storage (Talley et al., 1984). Certain studies on the free amino acid content in stored potato tubers have shown high values of amide as para gi ne and glutamine in tubers (Brierley et al., 1997). Amino acids content is associated with Millard reaction and acrylamide formation. Several studies have shown that acrylamide can be formed in Millard reaction from asparagine with carbonyl compounds (Stadler et al., 2002; Zyzak et al., 2003; De Wilde et al., 2005). The relationship among the quantity of reducing sugars, amino acids content and typical co50 M. Černá, S. Kráčmar lour of potato products was investigated by many researches (Roe et al., 1990; Brierley et al., 1996). Most studies in the fi eld of amino acids content in potato have only focused on the amount of free amino acids. So far, there have been only a few discussions about the total amino acids content in stored potato tubers. The purpose of the present study was to determine the infl uence of storage duration on the amino acids content, amino acids profi le and amino acid score in six potato varieties. MATERIALS AND METHODS Storage and sampling of potato tubers Potatoes (Solanum tuberosum L.) were grown in fi ve locations in the Czech Republic on the premises of AGRO-MĚŘÍN, a.s. Six potato cultivars with diff erent maturity class were selected for the experiment. Characteristics of the cultivars are shown in Tab. I. Winter barley (Hordeum vulgare) was used as a fore crop. In the autumn, manure was applied (45 t . ha−1) together with nitrogen (40.5 kg N.ha−1 for straw decomposition) and potassium fertilization. Plants were grown using a standard commercial practice and a uniform fertilization regime. Nitrogen, potassium and phosphorus fertilization, in doses 131 kg N.ha−1 (UREA Stabil), 162 kg K.ha−1 (K2O) and 72 kg P.ha−1 (P2O5), were used in the experiment. Tubers were harvested in the period from August till October 2008. Randomly selected tubers were stored in a stora ge house (Beskyd Fryčovice, a.s., CZ) with an automatic air-conditioning control. Potatoes were kept at 3–6 °C and 80% RH in 20-kilogram boxes. Sprout formation during the storage was suppressed by NEO-STOP L 300 treatment in the form of gas. Tubers were sampled for analysis at 4-week intervals, in total period of 16 weeks. Each month a sample consisting of two 20-kilogram boxes was sent to labo ra to ry analysis. About two kilograms of healthy tubers of each delivery were selected, thoroughly washed to remove soil particles, peeled and ground. All the samples were kept at −80 °C and then lyophilized at −4 °C, 12.156 Pa for 48 h (Christ Alpha1–4 LSC, Labicom, CZ) before the analysis. Total nitrogen content Total nitrogen was determined by the micro of Kjeldahl method by an automatic Pro-Nitro 130. Conversion factor of 6.25 was used for the calculation of the crude protein content. Chemical analyses were repeated fi ve times and the results were expressed on 100% dry matter basis (DM). Amino acids analysis To determine the fi een amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, histidine, lysine and arginine), 0.1 g of lyophilized tubers was weighted into screw-capped vial. Amount 15 ml of 6 mol . l−1 HCl was added into a vial and the solution was purged by argon for 1 min. The vial was consequently placed in a term-block (Labicom, CZ) which was heated to 115 °C and a sample was hydrolyzed for 23 hours. Then the vial was cooled down, HCl was removed by a rotary va cuum evaporator (RVO 400a, Ingos, CZ) and the ropy residue was quantitatively transferred into the 25 ml volumetric fl ask with sodium-citrate buff er (pH 2.2). For the assessment of the sulphur amino acids (methionine and cysteine), 0.7 g of lyophilized tubers was oxidized in hydrogen peroxide, formic acid and phenol. Oxidation was accomplished at 2 °C for 16 hours (Offi cial Journal of the European Union, 2009). The following procedure was the same as that of acid hy droly sis. The sample was fi ltered through a 0.45 μm fi lter before using the analyzer. For the assignment of amino acids, the ionic liquid chromatography with sodium-citrate buff ers, post-column ninhydrin derivatization and spectrophotometric detection (Amino Acid Analyzer AAA 400, Ingos, CZ) was applied. Each sample was repeated six times and each hydrolysate was analyzed twice. The results were expressed in 16 g N. I: Characteristics of potato samples Variety Maturing Boiling type Locality GPS Planting Harvest Angela VE B Žďár/Sázavou 49°33 ́17 ́ ́N; 15°54 ́20 ́ ́E 17/05 29/08 Princess VE A Krmelín 49°44 ́07 ́ ́N; 18°14 ́03 ́ ́E 30/05 10/09 Marabel E B Měřín 49°25 ́01 ́ ́N; 15°54 ́41 ́ ́E 20/05 15/08 Laura E B Paskov 49°43 ́50 ́ ́N; 18°17 ́51 ́ ́E 15/05 09/10 Milva SE AB Paskov 49°43 ́50 ́ ́N; 18°17 ́51 ́ ́E 10/05 04/09 Belana SE AB Petřvald 49°50 ́04 ́ ́N; 18°17 ́51 ́ ́E 12/05 04/10 VE – very early; E – early; SE – semi-early The eff ect of storage on the amino acids composition in potato tubers 51 All results were evaluated using the variation statistics, correlation matrices and regression functions were calculated according to Snedecor and Cochran (1967) using the statistical package Unistat (v. 5.5). RESULTS AND DISCUSSION The changes in the sum of nonessential (∑NEAA), essential amino acids (∑EAA) and total amino acids (∑AA) in potato tubers of diff erent varieties during the 16-week storage are presented in Tab. II. The results demonstrate that the storage duration has a signifi cant infl uence on the amino acids content in all potato varieties. At the beginning of the storage, the value of ∑AA moved in the range of 79.9–87.2 g/16 g N. The results have shown that Angela variety contained the maximum of total amino acids. A er 16-week storage of the potatoes, the total quantity of amino acids decreased approximately by 20%. The highest loss in the total amino acids content was observed in the potato variety Belana (28.4%), whereas the lowest reduction of ∑AA (8.7%) was recorded in Marabel variety. Both va rieties belong to early potato varieties. A considerable fall (27%) in ∑NEAA was found in samples of Princess variety, which was stored for 4 months. Minor reduction in ∑NEAA resulting from the storage duration was observed in Marabel variety. The highest amount of essential amino acids was found in tubers of early variety Angela. Semi-early varieties Marabel and Belana had comparable values of ∑EAA. From the data in Tab. II, it is apparent that the amino acid composition is signifi cantly infl uenced by potato variety. There exists a certain evidence that the stora ge time slightly eff ected the amino acids content in tuber of Marabel variety, whereas the value of the amino acids in Milva variety was markedly infl uenced by the storage duration. Tab. III shows the mean value of chemical score (CS) and essential amino acids index (EAAI) of the tested potato va rie ties. The chemical score with respect to the refe ren ce pattern (whole egg protein) was calculated for each cultivar. At the beginning and at the end of the storage, the mean value of the EAAI (con side ring all the essential amino acids except tryptophan) ranged from 54–66 and 42–57%, respectively. These results appear to be consistent with the previously published data (Sotelo et al., 1998; Galdón et al., 2010). The researches who used a reference protein (WHO/FAO) found the value of chemical score of amino acids between 42–53%. Further, it is known that potatoes contain a high amount of lysine which occurs rarely in plant proteins. The highest amount of lysine was determined in Marabel and Angela. At the beginning of the experiment, sulphuric amino acids (Cys+Met) were found as limiting amino acids in all the samples except Milva. It this variety, isoleucine was found as the fi rst amino acid limiting the protein nutrition value. A er the 16-week stora ge, Cys and Met were repeatedly detected as li miting acids in the tested varieties except Angela variety which had isoleucin as the fi rth and Cys and Met as the second limiting amino acids. Leucine was the second limiting amino acid for Milva and isoleuII: Eff ect of 4-month storage on the sum of nonessential, essential and total amino acids in potato tubers in g/16 g N Variety Angela Princess Marabel Belana Milva Laura Storage (month) 1 4 1 4 1 4 1 4 1 4 1 4 ∑NEAA 51.30 41.96 53.78 39.17 49.08 43.50 49.07 37.77 46.71 36.77 50.18 40.30 ∑EAA 35.87 29.60 29.56 24.89 34.75 33.02 34.89 22.43 33.21 24.40 32.61 26.82 ∑AA 87.16 71.56 83.34 64.06 83.83 76.52 83.96 60.20 79.92 61.17 82.79 67.18 III: Changes of Chemical score and Essential amino acids index in potato tubers during 4-month storage Index Variety Angela Princess Marabel Belana Milva Laura EAA 1 4 1 4 1 4 1 4 1 4 1 4 CS Thr 69.2 54.6 64.3 41.2 69.2 60.8 61.1 54.6 64.3 41.6 60.3 41.9 Val 80.5 63.2 68.5 61.0 74.9 71.4 74.5 63.2 68.5 53.4 74.9 59.9 Leu 63.8 52.8 49.3 43.3 61.0 66.3 65.1 52.8 49.3 43.7 63.2 43.9 Ile 55.4 44.6* 44.9 40.8 50.6 50.8 53.6 44.6 44.9* 39.0 51.3 41.8 Lys 75.4 58.8 61.5 47.0 74.0 72.9 66.0 58.8 61.5 51.1 67.5 50.7 Arg 67.1 57.4 51.1 52.2 67.7 51.3 80.1 57.4 51.1 48.7 54.0 68.9 His 63.1 53.2 61.7 53.5 66.6 64.5 62.7 53.2 61.7 50.2 61.2 58.9 Phe 67.7 54.9 55.8 45.5 66.1 65.6 65.7 54.9 55.8 45.6 62.5 48.1 ∑Cys+Met 52.2* 52.4 41.0* 37.1* 50.1* 43.1* 49.1* 38.4* 50.1 34.4* 44.7* 38.8* EAAI 66.1 53.8 54.1 44.4 61.0 56.9 60.5 55.8 54.1 42.4 57.5 47.6 CS – Chemical score EAAI – Essential amino acids index * – The fi rst limited amino acid 52 M. Černá, S. Kráčmar cine for the rest of the varieties. Suárez-López et al. (2006) have also found a very low level of sulfuric acids in the research that potato proteins content. Figures 1–6 compare the content of total amino acids (AA) and crude protein (CP) in diff erent potato varieties during the storage. At the beginning of the storage, the amount of CP was in the range from 8.9–11.9% of 100% DM. This fi nding is in an agreement with Galdón’s et al. (2010) results, where it is mentioned that crude protein content ranged from 1.46–2.38% of the fresh weight. Similar results are mentioned by other investigators (Blenkinsop et al., 2003). De Wilde’s et al. (2005) research shows that crude protein was fairly constant in tubers stored for 24 weeks. According to our results, longer stora ge time results in a decrease of the content of amino acids and the similar downward trend can be seen in crude protein. A er the 16-week sto ra ge, the amount of CP and AA decreased approx. by 35 and 18%, respectively. A rapid change in CP value (24%) was found in the samples of Belana variety. This variety showed the highest drop of total amino acids content (46%). On the contrary, a slight change in the amount of CP (10%) was observed in the tubers of Milva variety and in the lowest fall of AA content (29%) was found in Marabel variety. There exists certain evidence that the amino acids content and the quantity of crude protein are aff ected by the storage time and by the variety, but not the same way. Bártová et al. (2009) studied the eff ect of cultivar, locality and nitrogen fertilization on crude protein content in tubers of starch processing potato. Bártová et al. show that the most important factor affecting CP is the cultivar that participated in the total variability with approximately 34%.