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Effect of pH on phosphorylation of potato protein isolate
Food Chemistry 138 (2013) 2321–2326
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of pH on phosphorylation of potato protein isolate J. Miedzianka ⇑, A. Pe˛ksa ´ skiego Str. 37/41, 51-630 Wrocław, Poland Department of Food Storage and Technology, Faculty of Food Science, Wroclaw University of Environmental and Life Sciences, Chełmon
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Article history: Received 19 July 2012 Received in revised form 6 November 2012 Accepted 11 December 2012 Available online 29 December 2012 Keywords: Potato protein isolate Chemical phosphorylation Chemical composition Functional properties
a b s t r a c t Potato protein isolate (PPI) was phosphorylated with sodium trimetaphosphate (STMP) at ambient temperature and various reaction pH (5.2, 6.2, 8.0 and 10.5) to improve the functional properties without impairing the nutritional availability. Changes in chemical composition (total and coagulable protein content, ash and minerals content and amino acid composition), functional properties (protein solubility index, emulsifying activity and foaming capacity, water and oil absorption capacity) and phosphorus were determined. The chemical composition and functional properties of phosphorylated potato protein isolate (PP-PPI) were significantly different (p < 0.05). The PP-PPI at pH 5.2 was characterised by the highest content of all amino acids, whereas, PP-PPI under alkaline conditions (pH 10.5) caused decrease in these compounds. PP-PPI at pH 8.0 had the highest oil absorption capacity, emulsion activity and foam capacity, whereas, PP-PPI at pH 10.5 had the highest WAC. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The solubility of proteins and their properties are strongly affected by pH. Soluble fractions account for about 75–85% of proteins present in potatoes. This has been shown by different authors (Ahldén & Trägårdh, 1992; Ralet & Guéguen, 2000) most of soluble potato protein consist of 44,000 or 55,000 and 66,000 Da molar weight fractions which had an isoelectric point in the acidic range between pH 4.5 and 5.5. Other fractions, stated about 30% of the soluble potato protein were separated isoelectrically at pH values of approximately 6.0, 6.5, 7.2 and 7.8 and consist mainly of two protein fractions with molecular weight close to 20,000 or 14,000 Da. The recovery of proteins from potato fruit juice (PFJ) by heat coagulation, usually in low pH (ca 4.0–5.0) similar to processing on an industrial scale, led to the isolation of most protein fractions, however they are denaturated to an extreme extent. Although obtained by this method, a commercial potato protein isolate is characterised by a high content of total protein and low ash content and has weak functional properties such as: solubility, water and oil absorption capacity. Studies of authors (Ralet & Gueguen, 2000; van Koningsveld et al., 2002) show that solubility of potato proteins at pH > 5 is closely correlated with the ionic strength of the solution and the presence of unfolded tuberine. In the case where the ionic strength of slightly acidic or neutral potato protein solution was increasing, the destructive effects of high temperatures on the protein are significantly reduced. By increasing ionic strength of the solution to ⇑ Corresponding author. Tel.: +48 71 32057716. E-mail addresses: [email protected], [email protected] (J. Miedzianka). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.12.028
0.2 M/dm3, denaturation temperatures of the majority of potato protein fractions also increase and destruction of the secondary structure of proteins occurs prior to complete denaturation. This in turn can impact protein digestibility and functional properties as well as the susceptibility on chemical reactions. To increase the ionic strength of colloidal protein solution NaCl or CaCl2 solutions were used (Ralet & Gueguen, 2000; van Koningsveld et al., 2002). Functional properties of food proteins can be improved by some modifications, however, nutritional value may be affected. Among all chemical modifications, phosphorylation has been proven as an important and efficient method for improving the functional properties of food proteins like soybean (Hirotsuka & Taniguchi, 1984; Kunsheng, Yangyang, & Yunxia, 2007; Sung, Chen, Liu, & Su, 1983), dairy whey (Li, Enomoto, Ohki, Ohtomo, & Aoki, 2005), soy whey (Li, Enomoto, Hayashi, Zhao, & Aoki, 2010), ovoalbumin (Li et al., 2005), b-lactoglobulin (Woo, Creamer, & Richardson 1982), casein and lysozyme (Matheis, Penner, Feeney, & Whitaker, 1983), bovine serum albumin (Enomoto et al., 2008), egg white (Li, Ibrahim, Sugimoto, Hatta, & Aoki, 2004) and other protein sources. The chemical phosphorylation method is popular mainly because many phoshoproteins occur in nature. Many of them like casein in milk and ovoalbumin in egg white are used as food in human diet and food functional ingredients. The most popular phosphorylation method of food proteins is a chemical one (sulfatation) where chemicals like phosphorus oxychloride POCl3 (Matheis et al., 1983), phosphoric acid (Yoshikawa, Sasaki, & Chiba, 1981) and trisodium trimetaphosphate (STMP) (Sung et al., 1983) are used. The introduction of phosphoryl residues increases the negative charge and hydratation, and changes the functional properties of proteins (Damodaran & Paraf, 1997). Of the phosphorylating reagents
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tested, so far, only POCl3 and STMP may have proven to be economical and practical applicable. However, the chemical phosphorylation of food proteins may not be easily accepted by consumers mainly because of harsh reaction conditions, nonspecific chemical reagent and difficulty in removing the reagent from the final product (Matheis et al., 1983; Woo et al., 1982). Sodium trimetaphosphate (STMP) is an inorganic polyphosphate salt that serve as a chelating, starch-modifying and a boneimaging agent in foods. It is an approved food additive, and has been reported to have less toxic and fewer physiological effects than any other straight-chain polyphosphates. The polyphosphate anions found in STMP can bind calcium more tightly than sodium, therefore, exchange their sodium ions when calcium ions are present (Laningan, 2001). Nonenzymatic phosphorylation with STMP appears to be able to solve many of the problems associated with POCl3. However, further research is needed to determine the exact incorporation of covalently bound Pi. Sung et al. (1983) used STMP for phosphorylation of soybean protein under alkaline conditions (pH 10.5–12.5, 25–45 °C, for 3 h). The reaction was found to be dependent on pH, temperature and concentration. Also Woo et al. (1982) obtained phosphorylated bovine protein stable in the alkaline pH range. The object of this study was to chemically phosphorylate potato protein isolate with sodium trimetaphosphate at ambient temperature at various pH to improve the functional properties without impairing its nutritional availability. 2. Materials and methods 2.1. Materials Fresh potatoes were purchased from an experimental station from Pawłowice, Poland. Sodium trimetaphosphate (STMP) was obtained from Sigma–Aldrich, Poland. All chemicals used in the experiment were of analytical grade. 2.2. Preparation of potato protein isolate (PPI) Potato protein isolate was obtained by thermal coagulation with the use of CaCl2 in the concentrations of 0.04% in potato juice (van Koningsveld et al., 2002). Potatoes were washed thoroughly with water and cut into pieces which were immediately dipped in a solution of Na2SO4 + 7.5 g Na2S2O5/50 cm3 to prevent enzymatic browning. The potato pieces were ground in a domestic type juice extractor. The resulting juice was allowed to settle for 30 min and filtered using a double-layer cheese cloth. The suspension was adjusted to pH 7 using 1 M NaOH. 3% CaCl2 solution was added to the heated juice, and it was heated till 70 °C for 20 min. After cooling it down to the room temperature, the coagulated potato protein was separated from the supernatant by centrifugation at 5500g (Contifuge Stratos by Hereaus). The wet protein (paste) was rewashed thoroughly with water and afterwards it was freeze-dried. The potato protein isolate was stored at 25 °C for further analysis. 2.3. Preparation of phosphorylated potato protein isolate (PP-PPI) Phosphorylation of potato protein isolate was performed according to the method of Hirotsuka and Taniguchi (1984). To 10% solution of PPI was added STMP (in the amounts of 0.01 g of STMP/g of protein). The mixture was continuously stirred at room temperature for 1 h. During the phosphorylation, pH was maintained accordingly at 5.2; 6.2; 8.2 and 10.5 by the dropwise addition of 1 M NaOH or 1 M HCl. The phosphorylation reaction was completed within 30 min and the free phosphoric acid and other contaminants were removed from the solution by adding to
sample the distilled water till the conductation of liquid was similar to distilled water. Phosphorylated potato protein isolate was freeze-dried at the temperature of 20 °C. A control sample was treated in the same way but with no use of sodium trimetaphosphate. 2.4. Determination of phosphorus content of PP-PPI Phosphorus content was determined by the colorimetric method after dry-combustion of a microwave system for a sample preparation Mars 5 (CEM Corporation). P levels are the sum of endogenous P in a sample plus that incorporated by the chemical treatment (Krełowska-Kułas, 1993). 2.5. Determination of chemical composition of PPI and PP-PPI Total and coagulable protein content (protein was precipitated with the use of CuSO4 solution) were determined using Kjeldahl method based on AOAC (1996). Protein content was calculated by multiplying the nitrogen content by a factor 6.25. Samples for determining the ash content were heated gradually to 550 °C, and the residues were weighed. Fat content was determined by Soxhlet method (AOAC, 1996). Samples for determining the trace elements content were determined with the method of atomic adsorption spectrometry using a Varian AA240FS apparatus (Szczepaniak, 2004). 2.6. Determination of amino acid composition of PPI and PP-PPI Amino acid composition was determined by ion-exchange chromatography after 24 h hydrolysis with 6 N HCl at 110 °C was performed according to the method of Spackman, Stein, and Moore (1958). Hydrolysed amino acids were determined using AAA-400, INGOS, Czech Republic. As a detector photometer working with two wave length 440 and 570 nm was used. Column height was 350 3.7 mm and was packed with ion exchanger Ostion LG ANB, INGOS. Column temperature was kept at 40–70 °C and detector at 121 °C. Prepared samples were analysed using the ninhydrin method. Amino acid composition was expressed as grammes of amino acids per 16 g of N. 2.7. Measurement of functional properties 2.7.1. Protein solubility index (PSI) Protein solubility in distilled water was determined by the method described by Jackman and Yada (1988). Samples of protein were dispersed in distilled water and the slurries were transferred to a water bath with shaking for 1 h at room temperature. Then they were centrifuged at 5000g for 30 s. Protein content in the supernatants was determined by Kjeldahl method (AOAC, 1996). 2.7.2. Water absorption capacity (WAC) and oil absorption capacity (OAC) Water absorption capacity was estimated according to Sosulski (1962). The contents of solutions were mixed for 30 s in every 10 min using a glass rod and after mixing seven times, centrifuged at 5260g for 25 min (Rotofix 32A by Hettich). The supernatants were carefully decanted; the contents of the tube were allowed to drain at 50 °C for 25 min and weighed. Water holding capacity was expressed as grammes of water per gramme of preparation. Oil absorption capacity was estimated according to Lin and Humbert (1974). The protein suspensions with sunflower oil were mixed for 1 min. The samples were allowed to stand for 30 min. The protein–oil mixtures were centrifuged at 3000g (Rotofix 32A, Hettich) and the unabsorbed oil was carefully sampled with 10 cm3 calibrated pipette, and the volume was recorded. Oil
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binding capacity was expressed as millilitres of oil bound per gramme of preparation. 2.7.3. Emulsifying activity (EA) Emulsification activity was estimated by the method of Yasumatsu et al. (1972). The protein suspensions with added oil were homogenised at 10,000 rpm for 1 min. They were centrifuged at 3000g for 5 min. Emulsion activity was expressed as the ratio of height of emulsified layer in the tube to height of the total contents in the tube. 2.7.4. Foaming capacity (FC) Foaming capacity was assessed according to the method of Puski (1975). The protein suspensions were stirred for 1 min. The blends were immediately transferred into a graduated cylinder. The volumes were recorded before and after stirring. Foam capacity was expressed as the volume increased due to stirring. For determination the foam stability, changes in foam volume in the cylinder were recorded after 10 min of storage. Foam capacity was expressed as the ratio of volume after whipping to volume before whipping. 2.8. Statistical analysis The analysis of chemical composition and functional properties were performed in duplicate and in triplicate, respectively. Results were calculated on 5% moisture of the preparation. Data were subjected to one-way (differences between doses in each kind of a preparation) and two-way (differences between preparations of each dose) Duncan’s multiple range test of ANOVA (using Statistica 9.0 software). Significant differences were determined at the p 6 0.05 level. 3. Results and discussion 3.1. Chemical composition Chemical composition of PPI (a control sample) and PP-PPIs (preparations phosphorylated at different pHs) is presented in Table 1. The control and phosphorylated samples were characterised by comparable content of total (on average 96.32%) and coagulable (on average 90.46%) proteins in comparison with a preparation modified at pH 10.5 (88.87% and 81.15%, respectively). The extract rate of the protein isolate was ca. 9 g from 1 l of potato juice (own, unpublished results). A decrease of total protein content in the sample obtained at pH 10.5 may be attributed to introduction of phosphate groups onto polypeptide chains and the solubilisation under alkaline conditions (Chen, Lin, & Sung, 1984). Effect of various reaction pH on the total phosphorus content of PP-PPI is presented in Table 1. The higher phosphorus content was noted at pH 8.0 (13.67 mg/100 g) and 10.5 (12.96 mg/100 g) compared with other preparations. It can be explained that higher energy phosphate compounds could react with OH and NH2 (Woo et al., 1982) groups on the side chains of proteins. However, the
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free OH groups in the protein molecules showed higher activity only in the alkaline conditions at pH > 9 because of their low activity. Whereas the free NH2 groups, with higher reacting activity, could react in both neutral and alkaline conditions so that specific groups could react by controlling reacting pH (Kunsheng et al., 2007). Hirotsuka and Taniguchi (1984) and Li et al. (2004) noticed similar observations. They found that the level of phosphorus in modified potato protein isolate increased with an increase in pH levels. From the results, the rate of phosphorylation was rather low and may be caused by low concentrations used and/or low reactivities due to steric or electronic properties of the serine and threonine residues. The total ash and trace element contents of PPI and PP-PPIs are presented in Table 2. The PP-PPI at different pHs were characterised by higher content of total ash when compared with an unmodified sample. Whereas, potassium (K), calcium (Ca) and magnesium (Mg) contents of PPI were significantly higher than PP-PPIs. The K, Ca, Mg and Zn contents of PP-PPI also increased with increase in pH. The highest trace element contents were in PP-PPIs modified at alkaline pH (811.00 mg/100 g). Mineral composition of protein preparations differed with type of raw material, from which they were obtained, and with the coagulation method. Plant protein preparations are not considered as the source of mineral compounds, because of little quantity and limited accessibility due to presence of chelating complexes. 3.2. Amino acid composition As presented Table 3, preparations obtained at pH 5.2, 6.2 and 8.0 contained more essential amino acids (mainly leucine (Leu), lysine (Lys), methionine with cysteine (Met + Cys) and phenyloalanine with tyrosine (Phe + Tyr)) than PPI control and the one obtained at high alkaline pH (10.5). The preparation phosphorylated at pH 5.2 was characterised by the highest content of all amino acids (89.27 g/16gN), whereas, conducting the phosphorylation under alkaline conditions (pH 10.5) caused a decrease in these compounds (69.29 g/16gN). All modified products contained similar or even more essential amino acid than there were established for FAO protein standard (FAO/WHO Report., 2007), particularly Met + Cys and Phe + Tyr. Similar observations were observed by Sung (1982) who phosphorylated soy protein isolate under alkaline conditions without reducing its nutritional value. It is also worth underlining the increasing level of lysine content in comparison with PPI and a preparation phosphorylated at pH 10.5 by phosphate groups, may protect e-NH2 against an undesirable reaction such as protein–sugar interaction (Hirotsuka & Taniguchi, 1984). 3.3. Functional properties 3.3.1. Water and oil absorption capacity Water absorption capacity (WAC) and oil absorption capacity (OAC) of PPI and PP-PPI are presented in Fig. 1. The WAC of studied protein preparations depended on the reaction pH (Fig. 1A). The WAC of phosphorylated preparations increased with an increase pH as compared with PPI (4.76 g of water/g of preparation). The
Table 1 Characteristics of potato protein isolates subjected to the phosphorylation at various reaction pH. pH
Dry matter (%)
Total protein (N 6.25)
Coagulable protein
Content of phosphorus (mg/100 g)
PPI 5.2 6.2 8.0 10.5
96.62b ± 0.01 97.55a ± 0.41 97.14ab ± 0.20 97.15ab ± 0.34 89.71c ± 0.64
95.29b ± 1.21 95.58b ± 0.01 97.26a ± 0.08 97.13a ± 0.48 88.87c ± 0.21
86.24a ± 0.42 85.55c ± 0.99 95.11b ± 1.53 94.94bc ± 0.87 81.15d ± 1.20
– 10.97d ± 0.18 11.18c ± 0.22 13.67a ± 0.37 12.96b ± 0.11
a, b, c, d – the same letters in columns related to PP-PPIs mean homogenous groups.
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Table 2 Total ash and trace elements content of potato protein isolates subjected to the phosphorylation at various reaction pH. Trace elements pH
Total ash
Macro elements
Micro elements K
Ca
Mg [mg/100g]
Fe
Zn
Cu
92.69c,d ± 1.45 127.76c ± 3.50 167.08b ± 1.76 200.96a ± 2.91 91.37d ± 0.07
139.09a ± 2.02 15.84d ± 2.12 18.95d ± 0.14 25.45c ± 0.39 125.18b ± 1.34
462.08b ± 2.53 72.65c ± 10.80 66.03c ± 0.81 71.35c ± 0.58 525.56a ± 15.69
48.73b ± 0.04 3.40e ± 0.01 3.66d ± 0.01 4.30c ± 0.04 50.79a ± 0.08
7.64c ± 0.14 8.93a ± 0.01 6.97d ± 0.09 7.18d ± 0.13 8.43b ± 0.23
7.15b ± 0.04 3.84c ± 0.05 3.86c ± 0.09 4.06c ± 0.06 8.52a ± 0.13
1.11a ± 0.01 0.16b ± 0.05 0.15b ± 0.03 0.01c ± 0.01 1.16a ± 0.06
(%) PPI 5.2 6.2 8.0 10.5
3.26c ± 0.15 6.63b ± 0.78 5.84b ± 0.33 8.32a ± 0.47 8.53a ± 0.68
Total trace elements
Na
763.11b ± 2.34 232.59e ± 4.01 266.69d ± 0.09 313.31c ± 1.17 811.00a ± 2.01
a, b, c, d, e – the same letters in columns related to PP-PPIs mean homogenous groups.
Table 3 Amino acid composition of potato protein isolates subjected to the phosphorylation at various reaction pH. pH
Essential amino acids Ile
Total amino acids
Leu
Lys
Met +Cys
Val
Phe + Tyr
Thr
6.86b ± 0.08 8.99a ± 0.08 8.40a ± 0.12 8.84a ± 0.14 6.83ab ± 0.15 5.30
5.74c ± 0.17 7.57a ± 0.12 7.12b ± 0.05 7.58a ± 0.03 5.76c ± 0.07 4.50
3.95b ± 0.16 6.79a ± 0.10 3.55cd ± 0.17 3.37d ± 0.05 3.59c ± 0.18 2.21
4.45b ± 0.17 5.74a ± 0.11 5.33a ± 0.13 5.67a ± 0.16 4.26b ± 0.29 3.90
8.38c ± 0.16 10.90a ± 0.18 10.23b ± 0.14 10.93a ± 0.03 8.22c ± 0.16 3.81
3.91b ± 0.18 5.17a ± 0.22 4.91a ± 0.14 5.06a ± 0.28 4.00b ± 0.05 2.30
(g/16g N) PPI 5.2 6.2 8.0 10.5 Standard protein FAO 2007
3.49b ± 0.14 4.32a ± 0.20 4.02a ± 0.23 4.39a ± 0.11 3.41b ± 0.05 3.01
69.49c ± 1.44 89.27a ± 5.01 83.15b ± 0.34 88.52ab ± 0.03 69.29c ± 0.25
a, b, c, d – the same letters in columns related to PP-PPIs mean homogenous groups.
Fig. 1. Effect of various reaction pH on the water absorption capacity (A) and oil absorption capacity (B) of PPI and PP-PPIs. Data are mean of three determinations.
highest WAC was observed for PP-PPI at pH 8.0 (6.18 g of water/g of preparation) and at 10.5 (6.26 g of water/g of preparation). It can be explained by covalent attachment of anionic phosphate groups to polypeptide chains and its increase in net electronegativity. Increased water holding capacity of phosphorylated soy protein isolate was also observed by Sung (1982) who modified gluten with phosphoric acid. However, Ochiai-Yanagi, Miyauchi, Saio and Watanabe (1978) did not observe any increase in water-holding capacity by phosphorylation of soybean protein. Oil absorption capacity is of great importance because it reflects the emulsifying capacity, a desirable characteristic of products
such as mayonnaise. As it follows from Fig. 1B, OAC significantly increased only at pH 8.0 (4.79 cm3 of oil/g of preparation). Other samples were characterised by similar values of OAC. The high OAC of proteins is required in a ground meal formulation, meat replacers and extenders, doughnuts, baked goods, and soups (Li et al., 2010). 3.3.2. Protein solubility index The most important factor in the protein development is solubility as it is involved in influencing other functional properties. Effect of various reaction pH on the solubility properties of PPI and
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Fig. 2. Effect of various reaction pH on the protein solubility index of PPI and PP-PPIs. Data are mean of three determinations.
PP-PPI is presented in Fig. 2. It showed that the phosphorylation did not significantly change the solubility properties of PP-PPI at various reaction pH. Only preparation obtained under alkaline condition (pH 10.5) was characterised by the lowest solubility (19.62%) which may be attributed to more polymerisation of proteins (Matheis et al., 1983; Sitohy, Chobert, & Haertié, 1995) and occurrence of transverse-bridges (Matheis et al., 1983). Researchers indicated from the literature that chemical phosphorylation of food proteins increased the solubility and decreased the pI of proteins (Kunsheng et al., 2007).
3.3.3. Emulsifying activity and foaming capacity The effect of various reaction pH on the emulsifying activity of PPI and PP-PPI is presented in Fig. 3A. The PPI phosphorylated at pH 8.0 had the highest emulsion activity (3.79%) as compared with other preparations. This may be attributed to better amphiphilic balance and prevention of coalescence of oil droplets by the electrostatic repulsion force of the introduced negative charges of the phosphate groups (Li et al., 2005). Also, Saio and Watanabe (1978) tried to phosphorylate soybean protein but they did not bring about any significant modification of the protein used.
Fig. 3. Effect of various reaction pH on the emulsifying activity (A) and foam capacity (B) of PPI and PP-PPIs. Data are mean of three determinations.
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Whereas, Hirotsuka and Taniguchi (1984) showed that emulsifying activity increased with increasing phosphorus content. Researchers reported in the literature that emulsifying activity of food proteins were improved by phosphorylation using POCl3 (Sung et al., 1983; Woo et al., 1982) or in the presence of pyrophosphate (Li et al., 2004). Matheis et al. (1983) on the other hand reported that the emulsifying activity of phosphorylated casein with POCl3 decreased. The effect of various reaction pH on the foam capacity of PPI and PP-PPI is presented in Fig. 3B. It was observed that with an increase in reaction pH from 5.2 to 8.0, the foam capacity of PP-PPIs increased as compared with PPI (1.58%). Whereas, conducting the phosphorylation at pH 10.5 caused significant decrease in FC. Similar observations were noted by Sung (1982) who phosphorylated soy protein with STMP and obtained the phosphorylated soy protein preparations characterised by better foam expansion as compared with a commercial soy protein isolate and sodium caseinate. 4. Conclusions The present work describes the first investigation to obtain phosphorylated potato protein preparations by a chemical method under various pH. As it results from presented data, conducting the phosphorylation of potato protein isolate with STMP under slightly alkaline pH (8.0) improves the majority of functional properties (water and oil absorption capacity, emulsifying activity and foaming capacity). Since phosphorylated PPI has good functional properties even in the acid range, it may be useful for improving the texture, colour, taste and nutrition in acidic foods. There are necessary some experiments with animals to determined the digestion, absorption and utilisation of the amino acid of phosphorylated potato proteins by mammals, and to study the introduction of potentially toxic residues onto the proteins that may prevent their direct utilisation in foods. References Ahldén, I., & Trägårdh, G. (1992). A study of soluble proteins from four potato varieties used in the Swedish starch industry. Food Chemistry, 44, 113–118. AOAC. (1996). Official method of analysis (15th 261 ed.). Washington, DC: Association of Official Analytical Chemists. Chen, H. J., Lin, S. J., & Sung, H. Y. (1984). Enzymatic preparation of seasoning 50 nucleotides from baker’s yeast. Proceedings of the National Science Council, 8, 124–128. Damodaran, S., & Paraf, A. (1997). Food proteins and their applications. CRC Press, New York: CRC Press. Enomoto, H., Li, C.-P., Morizane, K., Ibrahim, H. R., Sugimoto, Y., Ohki, S., et al. (2008). Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate. Journal of Food Science, 73, C84–C91. FAO/WHO Report (2007). Energy and Protein Requirements. World Health Organization Technical Report. Series No. 935. Geneva.
Hirotsuka, M., & Taniguchi, H. (1984). Functionality and digestability of a highly phosphorylated soybean protein. Agricultural and Biological Chemistry, 48, 93–100. Jackman, R. L., & Yada, R. Y. (1988). Functional properties of whey-potato protein composite blends in a model system. Journal of Food Science, 53, 1427–1451. Krełowska-Kułas, M. (1993). Badanie jakos´ci produktów spoz_ ywczych. Warsaw: PWE. Kunsheng, Z., Yangyang, L., & Yunxia, R. (2007). Research on the phosphorylation of soy protein isolate with sodium tripoly phosphate. Journal of Food Engineering, 79, 1233–1237. Laningan, R. S. (2001). Final report on the safety assessment of sodium metaphosphate, sodium trimetaphosphate, and sodium hexametaphosphate. International Journal of Toxicology, 20, 75–89. Li, C.-P., Enomoto, H., Hayashi, Y., Zhao, H., & Aoki, T. (2010). Recent advances in phosphorylation of food proteins: a review. Food Science and Technology LWT, 43, 1295–1300. Li, C.-P., Enomoto, H., Ohki, S., Ohtomo, H., & Aoki, T. (2005). Improvement of functional properties of whey protein isolate through glycation and phosphorylation by dry heating. Journal of Dairy Science, 88, 4137–4145. Li, C.-P., Ibrahim, H. R., Sugimoto, Y., Hatta, H., & Aoki, T. (2004). Improvement of functional properties of egg white protein through phosphorylation by dry – heating in the presence of pyrophosphate. Journal of Agriculture and Food Chemistry, 52, 5752–5758. Lin, M. J. Y., & Humbert, E. S. (1974). Certain functional properties of sunflower meal products. Journal of Food Science, 39, 368–370. Matheis, G., Penner, M. H., Feeney, R. E., & Whitaker, J. R. (1983). Phosphorylation of casein and lysozyme by phosphorus oxychloride. Journal of Agriculture and Food Chemistry, 31, 379–387. Ochiai-Yanagi, S., Miyauchi, H., Saio, K., & Watanabe, T. (1978). Modified soybean protein with high water-holding capacity. Cereal Chemistry, 55, 157–167. Puski, G. (1975). Modification of functional properties of soy proteins by proteolytic enzyme treatment. Cereal Chemistry, 52, 655–662. Ralet, M.-C., & Guéguen, J. (2000). Fractionation of potato proteins: solubility, thermal coagulation and emulsifying properties. Food Science and Technology LWT, 33, 380–387. Saio, K., & Watanabe, T. (1978). Differences in functional properties of 7S and 11S soybean proteins. Journal of Texture Studies, 9, 135–157. Sitohy, M., Chobert, J.-M., & Haertié, T. (1995). Amino acid grafting of blactoglobulin mediated by phosphorus oxychloride. International Journal of Biological Macromolecules, 17, 269–272. Sosulski, F. W. (1962). The centrifuge method for determining flour absorption in hard red spring wheats. Cereal Chemistry, 39, 344–350. Spackman, D. H., Stein, W. H., & Moore, S. (1958). Automatic recording apparatus for use in the chromatography amino acid. Analytical Biochemistry, 30, 1190–1206. Sung, F. (1982). Chemical phosphorylation of food proteins by sodium trimetaphosphate. Food Science, 48, 716–719. Sung, H.-Y., Chen, H.-J., Liu, T.-Y., & Su, J.-C. (1983). Improvement of the functionalities of soy protein isolate through chemical phosphorylation. Journal of Food Science, 48, 716–721. Szczepaniak, W. (2004). Instrumental methods in chemical analysis (in Polish). Warsaw: PWN. Van Koningsveld, G., Gruppen, H., de Jongh, H. H. J., Wijngaards, G., van Boekel, M. A. J. S., Walstra, P., et al. (2002). Effects of pH and heat treatments on the structure and solubility of potato proteins in different preparations. Journal of Agriculture and Food Chemistry, 49, 4889–4897. Woo, S. L., Creamer, L. K., & Richardson, T. (1982). Chemical phosphorylation of bovine b-lactoglobulin. Journal of Agriculture and Food Chemistry, 30, 65–70. Yasumatsu, K., Sawada, K., Moritaka, S., Misaki, M., Toda, J., & Ishii, K. (1972). Whipping and emulsifying properties of soybean products. Agricultural Biological Chemistry, 36, 719–722. Yoshikawa, M., Sasaki, R., & Chiba, H. (1981). Effect of phosphorylation of bovine casein components on the properties related to casein micelle formation. Agricultural and Biological Chemistry, 44, 909–914.
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Effect of pH on phosphorylation of potato protein isolate J. Miedzianka ⇑, A. Pe˛ksa ´ skiego Str. 37/41, 51-630 Wrocław, Poland Department of Food Storage and Technology, Faculty of Food Science, Wroclaw University of Environmental and Life Sciences, Chełmon
a r t i c l e
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Article history: Received 19 July 2012 Received in revised form 6 November 2012 Accepted 11 December 2012 Available online 29 December 2012 Keywords: Potato protein isolate Chemical phosphorylation Chemical composition Functional properties
a b s t r a c t Potato protein isolate (PPI) was phosphorylated with sodium trimetaphosphate (STMP) at ambient temperature and various reaction pH (5.2, 6.2, 8.0 and 10.5) to improve the functional properties without impairing the nutritional availability. Changes in chemical composition (total and coagulable protein content, ash and minerals content and amino acid composition), functional properties (protein solubility index, emulsifying activity and foaming capacity, water and oil absorption capacity) and phosphorus were determined. The chemical composition and functional properties of phosphorylated potato protein isolate (PP-PPI) were significantly different (p < 0.05). The PP-PPI at pH 5.2 was characterised by the highest content of all amino acids, whereas, PP-PPI under alkaline conditions (pH 10.5) caused decrease in these compounds. PP-PPI at pH 8.0 had the highest oil absorption capacity, emulsion activity and foam capacity, whereas, PP-PPI at pH 10.5 had the highest WAC. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The solubility of proteins and their properties are strongly affected by pH. Soluble fractions account for about 75–85% of proteins present in potatoes. This has been shown by different authors (Ahldén & Trägårdh, 1992; Ralet & Guéguen, 2000) most of soluble potato protein consist of 44,000 or 55,000 and 66,000 Da molar weight fractions which had an isoelectric point in the acidic range between pH 4.5 and 5.5. Other fractions, stated about 30% of the soluble potato protein were separated isoelectrically at pH values of approximately 6.0, 6.5, 7.2 and 7.8 and consist mainly of two protein fractions with molecular weight close to 20,000 or 14,000 Da. The recovery of proteins from potato fruit juice (PFJ) by heat coagulation, usually in low pH (ca 4.0–5.0) similar to processing on an industrial scale, led to the isolation of most protein fractions, however they are denaturated to an extreme extent. Although obtained by this method, a commercial potato protein isolate is characterised by a high content of total protein and low ash content and has weak functional properties such as: solubility, water and oil absorption capacity. Studies of authors (Ralet & Gueguen, 2000; van Koningsveld et al., 2002) show that solubility of potato proteins at pH > 5 is closely correlated with the ionic strength of the solution and the presence of unfolded tuberine. In the case where the ionic strength of slightly acidic or neutral potato protein solution was increasing, the destructive effects of high temperatures on the protein are significantly reduced. By increasing ionic strength of the solution to ⇑ Corresponding author. Tel.: +48 71 32057716. E-mail addresses: [email protected], [email protected] (J. Miedzianka). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.12.028
0.2 M/dm3, denaturation temperatures of the majority of potato protein fractions also increase and destruction of the secondary structure of proteins occurs prior to complete denaturation. This in turn can impact protein digestibility and functional properties as well as the susceptibility on chemical reactions. To increase the ionic strength of colloidal protein solution NaCl or CaCl2 solutions were used (Ralet & Gueguen, 2000; van Koningsveld et al., 2002). Functional properties of food proteins can be improved by some modifications, however, nutritional value may be affected. Among all chemical modifications, phosphorylation has been proven as an important and efficient method for improving the functional properties of food proteins like soybean (Hirotsuka & Taniguchi, 1984; Kunsheng, Yangyang, & Yunxia, 2007; Sung, Chen, Liu, & Su, 1983), dairy whey (Li, Enomoto, Ohki, Ohtomo, & Aoki, 2005), soy whey (Li, Enomoto, Hayashi, Zhao, & Aoki, 2010), ovoalbumin (Li et al., 2005), b-lactoglobulin (Woo, Creamer, & Richardson 1982), casein and lysozyme (Matheis, Penner, Feeney, & Whitaker, 1983), bovine serum albumin (Enomoto et al., 2008), egg white (Li, Ibrahim, Sugimoto, Hatta, & Aoki, 2004) and other protein sources. The chemical phosphorylation method is popular mainly because many phoshoproteins occur in nature. Many of them like casein in milk and ovoalbumin in egg white are used as food in human diet and food functional ingredients. The most popular phosphorylation method of food proteins is a chemical one (sulfatation) where chemicals like phosphorus oxychloride POCl3 (Matheis et al., 1983), phosphoric acid (Yoshikawa, Sasaki, & Chiba, 1981) and trisodium trimetaphosphate (STMP) (Sung et al., 1983) are used. The introduction of phosphoryl residues increases the negative charge and hydratation, and changes the functional properties of proteins (Damodaran & Paraf, 1997). Of the phosphorylating reagents
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tested, so far, only POCl3 and STMP may have proven to be economical and practical applicable. However, the chemical phosphorylation of food proteins may not be easily accepted by consumers mainly because of harsh reaction conditions, nonspecific chemical reagent and difficulty in removing the reagent from the final product (Matheis et al., 1983; Woo et al., 1982). Sodium trimetaphosphate (STMP) is an inorganic polyphosphate salt that serve as a chelating, starch-modifying and a boneimaging agent in foods. It is an approved food additive, and has been reported to have less toxic and fewer physiological effects than any other straight-chain polyphosphates. The polyphosphate anions found in STMP can bind calcium more tightly than sodium, therefore, exchange their sodium ions when calcium ions are present (Laningan, 2001). Nonenzymatic phosphorylation with STMP appears to be able to solve many of the problems associated with POCl3. However, further research is needed to determine the exact incorporation of covalently bound Pi. Sung et al. (1983) used STMP for phosphorylation of soybean protein under alkaline conditions (pH 10.5–12.5, 25–45 °C, for 3 h). The reaction was found to be dependent on pH, temperature and concentration. Also Woo et al. (1982) obtained phosphorylated bovine protein stable in the alkaline pH range. The object of this study was to chemically phosphorylate potato protein isolate with sodium trimetaphosphate at ambient temperature at various pH to improve the functional properties without impairing its nutritional availability. 2. Materials and methods 2.1. Materials Fresh potatoes were purchased from an experimental station from Pawłowice, Poland. Sodium trimetaphosphate (STMP) was obtained from Sigma–Aldrich, Poland. All chemicals used in the experiment were of analytical grade. 2.2. Preparation of potato protein isolate (PPI) Potato protein isolate was obtained by thermal coagulation with the use of CaCl2 in the concentrations of 0.04% in potato juice (van Koningsveld et al., 2002). Potatoes were washed thoroughly with water and cut into pieces which were immediately dipped in a solution of Na2SO4 + 7.5 g Na2S2O5/50 cm3 to prevent enzymatic browning. The potato pieces were ground in a domestic type juice extractor. The resulting juice was allowed to settle for 30 min and filtered using a double-layer cheese cloth. The suspension was adjusted to pH 7 using 1 M NaOH. 3% CaCl2 solution was added to the heated juice, and it was heated till 70 °C for 20 min. After cooling it down to the room temperature, the coagulated potato protein was separated from the supernatant by centrifugation at 5500g (Contifuge Stratos by Hereaus). The wet protein (paste) was rewashed thoroughly with water and afterwards it was freeze-dried. The potato protein isolate was stored at 25 °C for further analysis. 2.3. Preparation of phosphorylated potato protein isolate (PP-PPI) Phosphorylation of potato protein isolate was performed according to the method of Hirotsuka and Taniguchi (1984). To 10% solution of PPI was added STMP (in the amounts of 0.01 g of STMP/g of protein). The mixture was continuously stirred at room temperature for 1 h. During the phosphorylation, pH was maintained accordingly at 5.2; 6.2; 8.2 and 10.5 by the dropwise addition of 1 M NaOH or 1 M HCl. The phosphorylation reaction was completed within 30 min and the free phosphoric acid and other contaminants were removed from the solution by adding to
sample the distilled water till the conductation of liquid was similar to distilled water. Phosphorylated potato protein isolate was freeze-dried at the temperature of 20 °C. A control sample was treated in the same way but with no use of sodium trimetaphosphate. 2.4. Determination of phosphorus content of PP-PPI Phosphorus content was determined by the colorimetric method after dry-combustion of a microwave system for a sample preparation Mars 5 (CEM Corporation). P levels are the sum of endogenous P in a sample plus that incorporated by the chemical treatment (Krełowska-Kułas, 1993). 2.5. Determination of chemical composition of PPI and PP-PPI Total and coagulable protein content (protein was precipitated with the use of CuSO4 solution) were determined using Kjeldahl method based on AOAC (1996). Protein content was calculated by multiplying the nitrogen content by a factor 6.25. Samples for determining the ash content were heated gradually to 550 °C, and the residues were weighed. Fat content was determined by Soxhlet method (AOAC, 1996). Samples for determining the trace elements content were determined with the method of atomic adsorption spectrometry using a Varian AA240FS apparatus (Szczepaniak, 2004). 2.6. Determination of amino acid composition of PPI and PP-PPI Amino acid composition was determined by ion-exchange chromatography after 24 h hydrolysis with 6 N HCl at 110 °C was performed according to the method of Spackman, Stein, and Moore (1958). Hydrolysed amino acids were determined using AAA-400, INGOS, Czech Republic. As a detector photometer working with two wave length 440 and 570 nm was used. Column height was 350 3.7 mm and was packed with ion exchanger Ostion LG ANB, INGOS. Column temperature was kept at 40–70 °C and detector at 121 °C. Prepared samples were analysed using the ninhydrin method. Amino acid composition was expressed as grammes of amino acids per 16 g of N. 2.7. Measurement of functional properties 2.7.1. Protein solubility index (PSI) Protein solubility in distilled water was determined by the method described by Jackman and Yada (1988). Samples of protein were dispersed in distilled water and the slurries were transferred to a water bath with shaking for 1 h at room temperature. Then they were centrifuged at 5000g for 30 s. Protein content in the supernatants was determined by Kjeldahl method (AOAC, 1996). 2.7.2. Water absorption capacity (WAC) and oil absorption capacity (OAC) Water absorption capacity was estimated according to Sosulski (1962). The contents of solutions were mixed for 30 s in every 10 min using a glass rod and after mixing seven times, centrifuged at 5260g for 25 min (Rotofix 32A by Hettich). The supernatants were carefully decanted; the contents of the tube were allowed to drain at 50 °C for 25 min and weighed. Water holding capacity was expressed as grammes of water per gramme of preparation. Oil absorption capacity was estimated according to Lin and Humbert (1974). The protein suspensions with sunflower oil were mixed for 1 min. The samples were allowed to stand for 30 min. The protein–oil mixtures were centrifuged at 3000g (Rotofix 32A, Hettich) and the unabsorbed oil was carefully sampled with 10 cm3 calibrated pipette, and the volume was recorded. Oil
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binding capacity was expressed as millilitres of oil bound per gramme of preparation. 2.7.3. Emulsifying activity (EA) Emulsification activity was estimated by the method of Yasumatsu et al. (1972). The protein suspensions with added oil were homogenised at 10,000 rpm for 1 min. They were centrifuged at 3000g for 5 min. Emulsion activity was expressed as the ratio of height of emulsified layer in the tube to height of the total contents in the tube. 2.7.4. Foaming capacity (FC) Foaming capacity was assessed according to the method of Puski (1975). The protein suspensions were stirred for 1 min. The blends were immediately transferred into a graduated cylinder. The volumes were recorded before and after stirring. Foam capacity was expressed as the volume increased due to stirring. For determination the foam stability, changes in foam volume in the cylinder were recorded after 10 min of storage. Foam capacity was expressed as the ratio of volume after whipping to volume before whipping. 2.8. Statistical analysis The analysis of chemical composition and functional properties were performed in duplicate and in triplicate, respectively. Results were calculated on 5% moisture of the preparation. Data were subjected to one-way (differences between doses in each kind of a preparation) and two-way (differences between preparations of each dose) Duncan’s multiple range test of ANOVA (using Statistica 9.0 software). Significant differences were determined at the p 6 0.05 level. 3. Results and discussion 3.1. Chemical composition Chemical composition of PPI (a control sample) and PP-PPIs (preparations phosphorylated at different pHs) is presented in Table 1. The control and phosphorylated samples were characterised by comparable content of total (on average 96.32%) and coagulable (on average 90.46%) proteins in comparison with a preparation modified at pH 10.5 (88.87% and 81.15%, respectively). The extract rate of the protein isolate was ca. 9 g from 1 l of potato juice (own, unpublished results). A decrease of total protein content in the sample obtained at pH 10.5 may be attributed to introduction of phosphate groups onto polypeptide chains and the solubilisation under alkaline conditions (Chen, Lin, & Sung, 1984). Effect of various reaction pH on the total phosphorus content of PP-PPI is presented in Table 1. The higher phosphorus content was noted at pH 8.0 (13.67 mg/100 g) and 10.5 (12.96 mg/100 g) compared with other preparations. It can be explained that higher energy phosphate compounds could react with OH and NH2 (Woo et al., 1982) groups on the side chains of proteins. However, the
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free OH groups in the protein molecules showed higher activity only in the alkaline conditions at pH > 9 because of their low activity. Whereas the free NH2 groups, with higher reacting activity, could react in both neutral and alkaline conditions so that specific groups could react by controlling reacting pH (Kunsheng et al., 2007). Hirotsuka and Taniguchi (1984) and Li et al. (2004) noticed similar observations. They found that the level of phosphorus in modified potato protein isolate increased with an increase in pH levels. From the results, the rate of phosphorylation was rather low and may be caused by low concentrations used and/or low reactivities due to steric or electronic properties of the serine and threonine residues. The total ash and trace element contents of PPI and PP-PPIs are presented in Table 2. The PP-PPI at different pHs were characterised by higher content of total ash when compared with an unmodified sample. Whereas, potassium (K), calcium (Ca) and magnesium (Mg) contents of PPI were significantly higher than PP-PPIs. The K, Ca, Mg and Zn contents of PP-PPI also increased with increase in pH. The highest trace element contents were in PP-PPIs modified at alkaline pH (811.00 mg/100 g). Mineral composition of protein preparations differed with type of raw material, from which they were obtained, and with the coagulation method. Plant protein preparations are not considered as the source of mineral compounds, because of little quantity and limited accessibility due to presence of chelating complexes. 3.2. Amino acid composition As presented Table 3, preparations obtained at pH 5.2, 6.2 and 8.0 contained more essential amino acids (mainly leucine (Leu), lysine (Lys), methionine with cysteine (Met + Cys) and phenyloalanine with tyrosine (Phe + Tyr)) than PPI control and the one obtained at high alkaline pH (10.5). The preparation phosphorylated at pH 5.2 was characterised by the highest content of all amino acids (89.27 g/16gN), whereas, conducting the phosphorylation under alkaline conditions (pH 10.5) caused a decrease in these compounds (69.29 g/16gN). All modified products contained similar or even more essential amino acid than there were established for FAO protein standard (FAO/WHO Report., 2007), particularly Met + Cys and Phe + Tyr. Similar observations were observed by Sung (1982) who phosphorylated soy protein isolate under alkaline conditions without reducing its nutritional value. It is also worth underlining the increasing level of lysine content in comparison with PPI and a preparation phosphorylated at pH 10.5 by phosphate groups, may protect e-NH2 against an undesirable reaction such as protein–sugar interaction (Hirotsuka & Taniguchi, 1984). 3.3. Functional properties 3.3.1. Water and oil absorption capacity Water absorption capacity (WAC) and oil absorption capacity (OAC) of PPI and PP-PPI are presented in Fig. 1. The WAC of studied protein preparations depended on the reaction pH (Fig. 1A). The WAC of phosphorylated preparations increased with an increase pH as compared with PPI (4.76 g of water/g of preparation). The
Table 1 Characteristics of potato protein isolates subjected to the phosphorylation at various reaction pH. pH
Dry matter (%)
Total protein (N 6.25)
Coagulable protein
Content of phosphorus (mg/100 g)
PPI 5.2 6.2 8.0 10.5
96.62b ± 0.01 97.55a ± 0.41 97.14ab ± 0.20 97.15ab ± 0.34 89.71c ± 0.64
95.29b ± 1.21 95.58b ± 0.01 97.26a ± 0.08 97.13a ± 0.48 88.87c ± 0.21
86.24a ± 0.42 85.55c ± 0.99 95.11b ± 1.53 94.94bc ± 0.87 81.15d ± 1.20
– 10.97d ± 0.18 11.18c ± 0.22 13.67a ± 0.37 12.96b ± 0.11
a, b, c, d – the same letters in columns related to PP-PPIs mean homogenous groups.
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Table 2 Total ash and trace elements content of potato protein isolates subjected to the phosphorylation at various reaction pH. Trace elements pH
Total ash
Macro elements
Micro elements K
Ca
Mg [mg/100g]
Fe
Zn
Cu
92.69c,d ± 1.45 127.76c ± 3.50 167.08b ± 1.76 200.96a ± 2.91 91.37d ± 0.07
139.09a ± 2.02 15.84d ± 2.12 18.95d ± 0.14 25.45c ± 0.39 125.18b ± 1.34
462.08b ± 2.53 72.65c ± 10.80 66.03c ± 0.81 71.35c ± 0.58 525.56a ± 15.69
48.73b ± 0.04 3.40e ± 0.01 3.66d ± 0.01 4.30c ± 0.04 50.79a ± 0.08
7.64c ± 0.14 8.93a ± 0.01 6.97d ± 0.09 7.18d ± 0.13 8.43b ± 0.23
7.15b ± 0.04 3.84c ± 0.05 3.86c ± 0.09 4.06c ± 0.06 8.52a ± 0.13
1.11a ± 0.01 0.16b ± 0.05 0.15b ± 0.03 0.01c ± 0.01 1.16a ± 0.06
(%) PPI 5.2 6.2 8.0 10.5
3.26c ± 0.15 6.63b ± 0.78 5.84b ± 0.33 8.32a ± 0.47 8.53a ± 0.68
Total trace elements
Na
763.11b ± 2.34 232.59e ± 4.01 266.69d ± 0.09 313.31c ± 1.17 811.00a ± 2.01
a, b, c, d, e – the same letters in columns related to PP-PPIs mean homogenous groups.
Table 3 Amino acid composition of potato protein isolates subjected to the phosphorylation at various reaction pH. pH
Essential amino acids Ile
Total amino acids
Leu
Lys
Met +Cys
Val
Phe + Tyr
Thr
6.86b ± 0.08 8.99a ± 0.08 8.40a ± 0.12 8.84a ± 0.14 6.83ab ± 0.15 5.30
5.74c ± 0.17 7.57a ± 0.12 7.12b ± 0.05 7.58a ± 0.03 5.76c ± 0.07 4.50
3.95b ± 0.16 6.79a ± 0.10 3.55cd ± 0.17 3.37d ± 0.05 3.59c ± 0.18 2.21
4.45b ± 0.17 5.74a ± 0.11 5.33a ± 0.13 5.67a ± 0.16 4.26b ± 0.29 3.90
8.38c ± 0.16 10.90a ± 0.18 10.23b ± 0.14 10.93a ± 0.03 8.22c ± 0.16 3.81
3.91b ± 0.18 5.17a ± 0.22 4.91a ± 0.14 5.06a ± 0.28 4.00b ± 0.05 2.30
(g/16g N) PPI 5.2 6.2 8.0 10.5 Standard protein FAO 2007
3.49b ± 0.14 4.32a ± 0.20 4.02a ± 0.23 4.39a ± 0.11 3.41b ± 0.05 3.01
69.49c ± 1.44 89.27a ± 5.01 83.15b ± 0.34 88.52ab ± 0.03 69.29c ± 0.25
a, b, c, d – the same letters in columns related to PP-PPIs mean homogenous groups.
Fig. 1. Effect of various reaction pH on the water absorption capacity (A) and oil absorption capacity (B) of PPI and PP-PPIs. Data are mean of three determinations.
highest WAC was observed for PP-PPI at pH 8.0 (6.18 g of water/g of preparation) and at 10.5 (6.26 g of water/g of preparation). It can be explained by covalent attachment of anionic phosphate groups to polypeptide chains and its increase in net electronegativity. Increased water holding capacity of phosphorylated soy protein isolate was also observed by Sung (1982) who modified gluten with phosphoric acid. However, Ochiai-Yanagi, Miyauchi, Saio and Watanabe (1978) did not observe any increase in water-holding capacity by phosphorylation of soybean protein. Oil absorption capacity is of great importance because it reflects the emulsifying capacity, a desirable characteristic of products
such as mayonnaise. As it follows from Fig. 1B, OAC significantly increased only at pH 8.0 (4.79 cm3 of oil/g of preparation). Other samples were characterised by similar values of OAC. The high OAC of proteins is required in a ground meal formulation, meat replacers and extenders, doughnuts, baked goods, and soups (Li et al., 2010). 3.3.2. Protein solubility index The most important factor in the protein development is solubility as it is involved in influencing other functional properties. Effect of various reaction pH on the solubility properties of PPI and
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Fig. 2. Effect of various reaction pH on the protein solubility index of PPI and PP-PPIs. Data are mean of three determinations.
PP-PPI is presented in Fig. 2. It showed that the phosphorylation did not significantly change the solubility properties of PP-PPI at various reaction pH. Only preparation obtained under alkaline condition (pH 10.5) was characterised by the lowest solubility (19.62%) which may be attributed to more polymerisation of proteins (Matheis et al., 1983; Sitohy, Chobert, & Haertié, 1995) and occurrence of transverse-bridges (Matheis et al., 1983). Researchers indicated from the literature that chemical phosphorylation of food proteins increased the solubility and decreased the pI of proteins (Kunsheng et al., 2007).
3.3.3. Emulsifying activity and foaming capacity The effect of various reaction pH on the emulsifying activity of PPI and PP-PPI is presented in Fig. 3A. The PPI phosphorylated at pH 8.0 had the highest emulsion activity (3.79%) as compared with other preparations. This may be attributed to better amphiphilic balance and prevention of coalescence of oil droplets by the electrostatic repulsion force of the introduced negative charges of the phosphate groups (Li et al., 2005). Also, Saio and Watanabe (1978) tried to phosphorylate soybean protein but they did not bring about any significant modification of the protein used.
Fig. 3. Effect of various reaction pH on the emulsifying activity (A) and foam capacity (B) of PPI and PP-PPIs. Data are mean of three determinations.
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Whereas, Hirotsuka and Taniguchi (1984) showed that emulsifying activity increased with increasing phosphorus content. Researchers reported in the literature that emulsifying activity of food proteins were improved by phosphorylation using POCl3 (Sung et al., 1983; Woo et al., 1982) or in the presence of pyrophosphate (Li et al., 2004). Matheis et al. (1983) on the other hand reported that the emulsifying activity of phosphorylated casein with POCl3 decreased. The effect of various reaction pH on the foam capacity of PPI and PP-PPI is presented in Fig. 3B. It was observed that with an increase in reaction pH from 5.2 to 8.0, the foam capacity of PP-PPIs increased as compared with PPI (1.58%). Whereas, conducting the phosphorylation at pH 10.5 caused significant decrease in FC. Similar observations were noted by Sung (1982) who phosphorylated soy protein with STMP and obtained the phosphorylated soy protein preparations characterised by better foam expansion as compared with a commercial soy protein isolate and sodium caseinate. 4. Conclusions The present work describes the first investigation to obtain phosphorylated potato protein preparations by a chemical method under various pH. As it results from presented data, conducting the phosphorylation of potato protein isolate with STMP under slightly alkaline pH (8.0) improves the majority of functional properties (water and oil absorption capacity, emulsifying activity and foaming capacity). Since phosphorylated PPI has good functional properties even in the acid range, it may be useful for improving the texture, colour, taste and nutrition in acidic foods. There are necessary some experiments with animals to determined the digestion, absorption and utilisation of the amino acid of phosphorylated potato proteins by mammals, and to study the introduction of potentially toxic residues onto the proteins that may prevent their direct utilisation in foods. References Ahldén, I., & Trägårdh, G. (1992). A study of soluble proteins from four potato varieties used in the Swedish starch industry. Food Chemistry, 44, 113–118. AOAC. (1996). Official method of analysis (15th 261 ed.). Washington, DC: Association of Official Analytical Chemists. Chen, H. J., Lin, S. J., & Sung, H. Y. (1984). Enzymatic preparation of seasoning 50 nucleotides from baker’s yeast. Proceedings of the National Science Council, 8, 124–128. Damodaran, S., & Paraf, A. (1997). Food proteins and their applications. CRC Press, New York: CRC Press. Enomoto, H., Li, C.-P., Morizane, K., Ibrahim, H. R., Sugimoto, Y., Ohki, S., et al. (2008). Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate. Journal of Food Science, 73, C84–C91. FAO/WHO Report (2007). Energy and Protein Requirements. World Health Organization Technical Report. Series No. 935. Geneva.
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