Nitrogen extractability and buffer capacity of defatted linseed ( Linum usitatissimum L.) flour

J. Sci. Food Agric. 1986,37, 199-205 Nitrogen Extractability and Buffer Capacity of Defatted Linseed (Linum usitatissimurn L.) Flour Dipak K. Dev, Erika Quensel and Rudolf Hansen Sektion Nahrungsgiiterwirtschaft und Lebensmitieltechnologie der Humboldt-Universitat zu Berlin, Neue Schonhauser Straj3e 20, 1020 Beriin, DDR (Manuscript received 20 February 1985) Defatted linseed flour was prepared from cold-pressed seed meal via hexane extraction of the residual oil, followed by removal of the major portion of the hulls through grinding and sieving (sieve size 0.25mm). The resulting flour had 50% protein on a dry basis compared with 40% contained by the whole seed on an oil-free dry basis. Nitrogen extractability of the defatted flour in water was not influenced by the length of the extraction period but an increased extraction was observed at higher so1id:liquid ratios up to the studied limit of 1:40. The smallest amount of nitrogen (20%) was extracted in the pH range 4.0-4.6 and the greatest (80%) at pH 12.0. Addition of NaCl (0.1-1.0) broadened the pH range of minimum nitrogen extractability and shifted it towards lower pH region. At higher concentrations (0.6 and 1.0 M) NaCl markedly increased nitrogen extractability in the pH range of 4.0 to 8.0. Precipitation of protein from an extract at pH 10.0 was maximum (77%) at pH 4.1. A higher buffer capacity of the flour was observed in the acidic medium (0.204 mmol HCI g- flour) than in the alkaline medium (0.096 mmol NaOH g- flour). Keywords: Linseed; nitrogen extractability; protein precipitability; buffer capacity. 1. Introduction Linseed is a potential source of protein, mucilage and dietary fibre. Its protein content ranges from 36 to 45% (on an oil-free dry basis) depending on variety and growing location. The seed is usually pressed in an expeller to remove a major part of the oil and the pressed cake is widely used as animal feed. A survey of oilseed processing in India has shown that about 70% of linseed is crushed in mills, compared with 17% of sesame, and in both cases the remainder is processed in cottage industry units, popularly known as ghani. It has been suggested that in India protein from linseed might be more expensive than groundnut and rapeseed protein but it would be much cheaper than that from sesame and other oleagenous sorces. The feasibility of preparing an attractive protein isolate from linseed with useful nutritional qualities has been demonstrated.6 The present paper describes the effect of extraction parameters such as pH, ionic strength, so1id:liquid (S:L) ratio and the time of extraction on the nitrogen extractability and the buffer capacity of defatted linseed flour. 2. Experimental 2.1. Materials Whole seed as well as cold-pressed linseed meal was provided by VEB Erfurter Oil Mill, Erfurt, German Democratic Republic. The meal was packed in polyethylene bags and stored in a refrigerator before conducting the experiments. Present address: College of Agricultural Technology, Marathwada Agricultural University, Parbhani-431402, Maharash- tra State, India. 199 200 D. K. Dev et d. 2.2. Preparation of defatted flour The residual oil from cold-pressed linseed meal was extracted with cold hexane at room temperature in a laboratory extractor. The solvent was removed from the extracted meal in a vacuum dryer at 40C for 6-43 h, ground in a coffee grinder at high speed and passed through a 0.25 mm sieve to obtain defatted flour. 2.3. Proximate composition Whole seed was finely ground in a coffee grinder before proximate analysis. Defatted flour was also analysed. Moisture and ash were determined by AOAC methods. Crude fat was determined by Soxhlet distillation with petroleum ether (b.p. 4C60C) for 6-8 h. Total nitrogen content was estimated by the semi-microKjeldah1 procedure and crude protein was calculated as Nx6.25. Crude fibre was estimated by a modified Scharrer and Kiirschner method.* 2.4. Nitrogen extractibality The influence of pH, ionic strength, S:L ratio and extraction time on the nitrogen extractability of defatted flour was investigated as shown in Table 1. In each experiment one parameter was varied and the other was held constant. Table 1. Experimental plan for studying the influence of extraction parameters on the nitrogen extractability of defatted linseed flour Experiment Parameter 1 2 3 4 S:L ratio (g:ml) 1:tO 1:20 1:20 120 120 1:30 1:40 Extraction 30 15, 30, 30 30 time (min) 60, 120 PH as obtained in the 2-12 2-12 aqueous suspension Ionic strength - - - 0.1; 0.3; (nNaCI) 0.6; 1.0 All extractions were at ambient temperature (ca22C) with frequent stirring using 1 g flour. Experiments 1, 2 and 3 were conducted using distilled water while experiment 4 with NaCl solutions of varying molar strength. In experiments where the pH was varied, the suspension was adjusted to the desired pH using 0.5 or 0.3 M HC1 or NaOH. The increase in volume as a result of pH-adjustment was noted and accounted for in all subsequent calculations. After each extraction, the suspension was centrifuged (2500xg for 30 min) at room temperature and the supernatant filtered. Nitrogen was determined by a semi-microKjeldah1 method by digesting 2 ml supernatant, and nitrogen extractability expressed as the percentage nitrogen extracted from the original defatted flour, assuming full recovery of the total volume. All extractions and determinations were performed in duplicate. 2.5. Protein precipitability Precipitability of protein was investigated by the method described by Taha et a1.9 with minor modifactions. Flour (log) was dispersed in 200ml distilled water and the pH of the suspension was adjusted to pH 10.0 using 0.5 NaOH. After extraction for 30 min with frequent stirring, the suspension was centrifuged and the supernatant filtered as described above. Aliquots (20 ml) of the filtrate were taken in graduated centrifuge tubes and adjusted to the desired pH levels ranging from 3.5 to 5.5 using 0.5 HCl. After centrifuging (2500Xg for 30 min), the volume of Nitrogen extractability and buffer capacity of linseed flour 201 the supernatant in each tube was noted. The overall yield of protein precipitation was calculated as V1N1- V2N2 VlNl x 100% where V, and V2 are the volumes of the aliquots before and after precipitation and Nl and N2 are mg nitrogen in 1 ml of V1 and V2 respectively. 2.6. Buffer capacity Flour (25 g) was dispersed in 500 ml distilled water and the dispersion divided into two portions of equal volume. To one portion was added known volumes of 0.1 NaOH and to the other 0.2 HCl and the corresponding changes in pH in both acid and alkaline ranges were noted. The amount of HCI or NaOH added was plotted against pH and the buffer capacities in either ranges were expressed as the mean values of mmol HCL or NaOH per g flour required to bring about a change in pH by one unit. 3. Results and discussion 3.1. Proximate composition The proximate composition of defatted linseed flour and that of whole seed, expressed on oil-free dry basis for comparison are shown in Table 2. The values are generally comparable with those reported in the literatre. Table 2. Proximate composition of linseed and defatted linseed flour prepared from cold-pressed meal Defatted flour, Dry basis Oil-free dry basis dry basis Whole seed Component (/.I (%I (%I The seed kernel Crude protein 24.4 39.7 50.1 Crude fat 128.6 - traces Ash 3.9 6.4 6.9 Crude fibre 6.1 9.9 5.4 N-free Extract 27.1 44.0 37.7 (Nx6.25) (by difference) constitutes about 56 to 70% of linseed. According to Sosulski and Cadden I the hull fraction of linseed is about 30% protein. However, as most of the oil is present in the kernel, defatting gives the hulls a low protein fraction compared with the kernel. Thus rejection of a major portion of the hulls through sieving the ground meal leads to defatted flour having a considerably higher protein content compared with the whole seed expressed on oil-free dry basis. The defatted flour has less crude fibre than the whole seed since a substantial portion of crude fibre is removed with the hulls. 3.2. Influence of extraction parameters on nitrogen extractibility 3.2.1. Extraction time and S:L ratio Nitrogen extractability data in distilled water at the pH as obtained in the normal suspension are shown in Figure 1. The length of the extraction period had very little influence on nitrogen extractability which remained practically constant with longer extraction times. However, variation of S:L ratio had a pronounced effect on the percent nitrogen extracted from the flour. 202 D. K. Dev et al. Extraction time (rnin) -Go Figure 1. Effect of extraction time and solid-liquid (S:L) ratio on nitrogen extractability of dcfatted linseed flour: U, extraction time; e-O, S:L I I I I ratio. 1:lO 1.20 1.30 1:40 S:L ratio (g: mll More and more nitrogenous material was extracted with progressively higher amounts of solvents. These observations are consistent with those of Gheysuddin et al. on sunflower and Rivas et al.I3 on sesame seed flour. Rtkowski reported a slight decrease in nitrogen extractability of rapeseed when the time of extraction extended beyond 30 min. Rivas et al.I3 however, reported a decrease in nitrogen extractability when a large excess of solvent was employed. These authors suggested that this was probably the result of lowered ionic strength because the flour had a high ash content. 3.2.2. pH and ionic strength The nitrogen extractability from defatted linseed flour in distilled water as well as in NaCl solutions of varying ionic strength within the pH range of 2-12 is illustrated in Figure 2. The broad pattern of nitrogen extractability at varying pH and ionic strength is comparable with those reported previously for linseed, 15, l6 and other defatted oilseed meals.*, l3 In water, nitrogen extractability increased steadily on both sides of the isoelectric range of the protein being extracted. This range appears to be roughly 4.M.6 where the extractability curve exhibits a minimum. Minimum extractability of nitrogenous matter from oil-free linseed meal was observed by Smith et al! at pH 3.8 and by Painter and Nesbitt16 at pH 3.5-4.0. Madhusudhan and Singh, however, recently reported a much broader pH range of least nitrogen solubility (pH 3.M.O) for demucilaged, defatted and dehulled linseed meal. These differences apparently are due to varying degrees of protein denaturation during the preparation of defatted meals. About 20% nitrogenous matter was extracted at isoelectric pH region (Figure 2). This is in agreement with other observations on linseed meals., l6 The nitrogen extractability increased with increasing acidity and reached about 40% at pH 2.0. Above pH 4.6 nitrogen extractability increased steadily until at pH 12.0 about 80% of the flour nitrogen was extracted. Sosulski and Bakal,4 however, could extract 91-96% of the total nitrogen from whole seed defatted meal employing 0.2% NaOH solution. At strong alkaline pH values such as those above pH 10.5, a levelling tendency with regard to nitrogen extractability is evident (Figure 2). Also, during extraction of proteins at alkaline pH Nitrogen extractability and buffer capacity of linseed flour 203 the adverse effects of alkali on proteins should not be overlooked. It is known that alkaline treatment of proteins at elevated temperatures results in a loss of lysine and cystine and formation of new amino acids such as lysinoalanine, ornithoalanine, beta-amino-alanine, lanthinine which are considered to be nutritionally detrimental. Lysinoalanine and lanthionine are also formed by heat treatment at neutral pH. Aymard et al. reported that defatted linseed, rapeseed and soya oilseed cakes, subjected to various heat treatments, contain 1.4-1.5 mg free lysinoalanine and 0.1-0.3 mg lanthionine per g protein. These observations underline the need for a compromise between protein yield and quality while attempting protein isolation through alkaline extraction. For the preparation of a protein isolate from defatted linseed meal the extraction could probably be made at a maximum pH of 10.0 at room temperature. NaCl widened the isoelectric range of linseed proteins shifting it simultaneously towards the more acidic side (Figure 2). At all four concentrations of NaCl, the percentage nitrogen extracted was rather low up to pH 4.0, about 25% on an average. However, above this pH, the nitrogen extractability registered a sharp rise at higher concentrations of NaCl(0.6 and 1 .O M). At 0.1 and 0.3 NaCl concentrations, however, extractability increased gradually. In general, addition of NaCl increased nitrogen extractability, particularly in the pH range 4.0-8.0. Maximum extractability of nitrogen was found to be about 76% at pH 8.0 in 1.0 NaCl. At this salt concentration further increase of pH extracted less nitrogen. At 0.6 M NaCl concentration and above pH 8.5 and at lower concentrations and above pH 10.0, nitrogen extractability levelled off gradually. The overall observations are similar to those described previously for linseed and sesame.13 90 80 70 - - 8 6C B ? ;i 50 c 0 c Cm P 3 4c 3c 2c IC 3 4 5 6 7 8 9 10 I1 12 DH Figure 2. Effect of pH and NaCl concentration on nitrogen extractability of defatted linseed flour. u, distilled water; U, 0.1 NaC1; 0-4, 0.3 NaC1; O., 0.6NaC1; O-.-.-o, 1.OM NaCl. 204 D. K. Dev et al. 6ot 50 L 3.5 4.0 4.5 5.0 5.5 PH Figure 3. Precipitability of protein from an alkaline extract (pH 10.0) from defatted linseed flour in the isoelectric pH region. 3.3. Protein precipitation In keeping with the pattern of minimum nitrogen extractability in water, the maximum precipitation of protein occurred in the pH range of 4.0-4.5; the peak was at pH 4.1 (Figure 3). About 77% of the extracted protein could be precipitated at this pH. The percentage protein precipitated decreased at progressively higher pH values and as acidity increased. Smith et al. l5 observed a maximum precipitation of dispersed linseed protein at a pH of 5.1 where about 21% of the nitrogenous matter remained soluble. 3t 21 I I I I I I J 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 rnrnol HCI or NaOH g- flow Figure 4. Buffer capacity of defatted linseed flour. a=0.204mmol HCI per g flour per unit pH; b=0.096mmol NaOH per g flour per unit pH. Nitrogen extractability and buffer capacity of linseed flour 205 3.4. Buffer capacity A 120 (S:Lj dispersion of defatted linseed flour in water had a pH of 6.3. Adding HCl or NaOH brought about changes in pH of the dispersion in proportion to the amount of HCI or NaOH added (Figure 4). At the alkaline pH, within the range of 6.3 to 11.5, an average of 0.096mmol NaOH per g flour was required in order to change the pH by one unit. To effect the same change at the acidic pH within the range of 6.3 to 3.9, 0.204mmol HCl per g flour was required. This indicates a markedly higher buffer capacity of the defatted flour in the acidic medium than in alkaline medium. Similar observations have been made by Rutkowski14 on rapeseed meal. During extraction of protein for preparation isolate, if pH 10.0 is used, the amount of NaOH required would roughly be 0.065 mmol per g flour. Acknowledgement The authors thank Mrs M. Seidenstucker for technical assistance. References 1. Dorrell, D. G. Flaxseed research in Canada. Fefte, Seifen, Ansfrichmifief 1975, 77, 258-260. 2. Annual Survey of Industries, 1965. CSO. Government of India, 1977,2, p. 125. 3. Singh, N. Linseed as a protein source. Indian Food Packer 1979. 33, 54-57. 4. Sosulski, F. W.; Bakel, A. Isolated proteins from rapeseed, flax and sunflower meals. Can. Insf. Food Technol. J. 1969, 2, 2b32. 5. Sosulski. F. W.: Sanvar. G. Amino acid composition of oilseed meals and protein isolates. Can. Znsr. Food Sci. Technol. 1. 1973, 6, 1-5. 6. Sarwar. G.; Sosulski, f. W.; Bell, J. M. Nutritional evaluation of oilseed meals and protein isolates by mice. Can. Inst. Food Sci. Technol. 1973, 6, 17-21. 7. A.O.A