Extraction of amino acids from beet juice. Emad M. E. Byuomi 1 Azza M. M. Ali 2 and Mahmoud A. Ghandour 3

Transcription

1 Extraction of amino acids from beet juice Emad M. E. Byuomi 1 Azza M. M. Ali 2 and Mahmoud A. Ghandour 3 1 Ph.D., Manager of Quality Control and Environment, Faiyum Sugar Company. 2 Professor of Analytical Chemistry, Faculty of Science, Assuit University, Assuit. 3 Professor of Analytical Chemistry, Faculty of Science, Assuit University, Assuit. Abstract: The amino acids in beet juice are scarcely studied and there are few contradictory reports dealing with the number and concentration of the amino acids therein. Amino acids added to ration to increase the nutritional value. It thought that amino acids could be separate from juice using chromatographic techniques. To separate the amino acids from inorganic salts and other organic substances (viz, sugars and colloidal matter) in the samples, a small bed (15 cm high 0.3 cm dia) of Amberjet-1200 cation exchange resin is used and then the following processes were done: Sample preparation, sample pretreatment, removal of neutral (and anionic) components in liquid samples, color removal, sample separation and sample identification ( HPLC and amino acids analyzer). The total amino acids were found to be ~ 5.44% (W/W) in beet juice. Amino acids analyses were done to separate them qualitatively and then a total of 20 different amino acid were found to be: Aspartic acid, serine, glutamic acid, glycine, valine, methionine, leucine, tyrosine, cystine, carnosine, alpha-amino adipic acid, alanine, beta-alanine, phenylalanine, beta-amino-isobutyric acid, gama-amino-n-butyric acid, histidine, tryptophan, lysine, arginin. The protein quality of beet juice is rather high, because of the high figures of essential amino acids, which were extracted from beet juice, as glutamic acid, valine, methionine, leucine, cystine, phenylalanine, histidine, tryptophan, lysine and agrinine. Application of the developed method is useful with respect to a better understanding of the background of the amino acid composition of sugar beets and the fate of amino acids along the sugar manufacturing process. 1 TL2.1/

2 This preliminary investigation will give an idea of the utilization of ion exchange resins, which is already present in all beet sugar companies (decalcification unit), to extract amino acids from juice to license this project in Egypt and evaluation of another Integrated Industries namely the extraction of amino acids from juice to be added as a concentrate nutrient to the beet pulp to increase its nutritional value, or to be used as a feed ingredient. Introduction The values of the deionization plant nonsucrose and amino acid eliminations by ion exchange resins were illustrated in Table 1. The juice treated was a 30 Brix, purity, mixture of intermediate green syrup and thin juice [1]. Table 1: Ion exchange eliminations Run Influent Effluent Percent nonsugars eliminated Total amino (N)/100 Sug Calcium /100 Sugar Sodium / 100 Sugar Potassium / 100 Sugar The chemical composition of beet juice shows wide variation. Its composition influenced by factors such as soil type, ambient temperature, moisture, season of production, variety, and production practices at a particular processing plant. Consequently, considerable variation found in nutrient content, color, viscosity and total sugar content. The composition data reflect these differences since these figures were compiled from analysis presented in several publications, where the ranges of crude ash was , crude protein and crude fibre % [2]. The beet juice contains significant levels of crude protein (N x 6.25). In addition, the nitrogenous materials present consist mainly of non-protein nitrogen compounds include amides, albuminoids, amino acids and other simple nitrogenous compounds. The effect of soil type on nutrient content illustrated that juices produced from beet grown on organic soils contained 5-6% amino acids as compared to 3% for molasses from mineral soils [3]. A new method for amino acid analysis was developed to improve the knowledge on the causes for both the variation in the amino acid composition of sugar beet and their absolute level. As a first example of the application of the analysis method, the total amino acids of one variety of 2 TL2.1/

3 sugar beet, named Auris, which grown on different soil types was clay soil 21.7, sand soil 27.5 and high peat soil 27.9 mmol/kg beets. The first relatively complete identification of the amino acids in beet juice was achieved to indicate that the following ten substances were present in beet: asparagine, aspartic acid, glutamic acid, serine, glycine, alanine, γ- amino butyric acid, lysine, valine and leucine or isomers [5,6]. The quality of a protein is related to its amino acid content. High quality proteins have a good balance of the essential amino acids. Poor quality proteins are deficient in amount or balance of the essential amino acids. When feeding non-ruminant animals, the amino acid content of the protein is of greater importance than the percent of protein present in the feed [7]. Essential amino acids those acids which must be provided in the ration of non-ruminant animals because the animals cannot synthesize them fast enough to meet their needs. Ruminant animals can generally synthesize the essential amino acids by rumen microbial action. Non-essential amino acids are synthesized in the body from other amino acids and therefore, do not have to be provided in the ration. There were 10 essential and 13 nonessential for swine and 14 for poultry [8]. Most amino acids produced by the hydrolysis of various protein sources. These may be plant, animal, or microorganism derived. A number of amino acids produce by fermentation. The fermentation may take place on a culture media composed of grains, sugar, juice, yeast, or other biological material. The culture media may also be composed of petrochemicals, such as paraffin, and synthetic nutrients such as ammonium chloride, ammonium nitrate, and potassium phosphate [9]. This paper describes the method development to extract amino acids and protein from beet juice using the already present ion exchange bed chromatography system in beet sugar companies (decalcification plant). This would be a natural method of amino acids production to be added as a concentrate nutrient to the beet pulp to increase its nutritional value and then fed to animals as a feed ingredient. Materials and Methods The present investigation was conducted at Faiyum Sugar Company, during the two successive beet seasons of 2004/05 and 2006/07 to evaluate the extraction of amino acids and protein from beet juice using ion exchange bed chromatography technique. 3 TL2.1/

4 The separation process designated as an ion exclusion separation, in the course of this study, several separation mechanisms were encountered. These include: 1- Ion exclusion separation, takes advantage that charged species (cations and anions) diffuse into the ionic matrix of ion exchange resin beds are more difficulty than small neutral molecules such as disaccharides and monosaccharides. 2- Size exclusion, which reflects the lower rate of diffusion of large molecules into the small pores of the resin matrix. As might be expected, these effects can both operate in varying degrees depending on the size and ionic nature of the species under consideration. However the principle of ion exchange separation of amino acids on a cation exchange resin are essentially based on: 1- Acid-base properties, i.e. charge of the amino acids; 2- Relative polarity of (neutral) amino acids; 3- Non-ionic, hydrophobic interactions with the styrene backbone of the resin. Experimental procedures for extracting of amino acids from juice using the pretreatment column filled with AMBERJET 1200 NA cation exchange resin A- Sample preparation 1- Weigh 25 g of the juice extracted from beet slicing by the warm digestion at 70 o C and transfer it into a wide-neck 200 ml volumetric flask using demineralised water. 2- Made level up till 2 ml below the 200 ml mark and the content is mixed by hand shaking. 3- Cool the sample to the room temperature. 4- Fill the flask exactly to the mark with demineralised water. 5- Shake the content of the flask to be homogenized. 6- Filter through a folded filter paper. The clear filtrate obtained after the warm digestion further subjected to the sample pretreatment described below. The filtrate solution can be stored at - 20 C. 4 TL2.1/

5 B- Sample pretreatment Due to the ionic character of amino acids they can be separated from neutral sugar by trapping them on a cation exchange resin. The small column (15 cm high 0.3 cm dia) filled with Amberjet 1200 NA cation exchange resin in the sodium form are very useful for that purpose. The ph of the sample solution must be first adjusted well below the isoelectric point of the basic amino acids, i.e. ph 8. The sample pretreatment procedure, which has specially developed for this purpose is described below. 1- Conditioning of column 1- Shake the column by hand to loosen the resin, remove the top cap and flush resin particles back into the column with demineralised water. 2- Put the column on top of a 100 ml flask and let the resin settle. 3- Open the cap at the bottom-outlet of column and remove the excess water from the column by drainage; prevent that the liquid level comes below that of the resin by closing the outlet with the cap. 4- Fill the column till the upper-rim with 0.1M NaOH and flush the resin; close the outlet with the cap when the liquid level is just above that of the resin. 2- Removal of neutral (and anionic) components in liquid samples 1- Defrost the filtrate sample and filter it through a 0.45 mm membrane. 2- Pour exactly 5 ml of the sample on the tops of the resins of column. 3- Alkalify the column by a subsequent addition of 1 ml 1M NaOH. 4- Stir the liquid above the resins gently by using a small plastic spoon and clean it by washing it with 0.1M NaOH. 5- Open the cap at the bottom-outlet of the column after settling of the resin (at a rate of about 1 hour) and drain the excess liquid from the column; subsequently, flush the resin with 1 M NaOH for about 1.5 hour at 0.2 ml/min. 6- Place the column on top of a 25 ml volumetric flask after weighing (X1g). 7- Add ammonia buffer solution (conc. 2%) to the columns in order to elute the amino acids. 5 TL2.1/

6 8- Make up a 25 ml volumetric flask to the mark by flushing the resin with the ammonia buffer solution: the collected, purified solution contains the amino acids, which 5 times diluted compared to the original extract from the warm juice digestion, which is further subjected to the sample decolorization. 3- Regeneration of column In principle the resin in the column can be used several times, when it is regenerated after the sample pretreatment by flushing it with 2-3 bed volumes of 1M NaOH and a subsequent washing with about 15 ml demineralised water. C- Color removal The active carbon particles was washed with water and then the purified solution passed through the column (15 cm high 0.3 cm dia) filled by granulated active carbon at a rate of about 0.5 hour (the amount used represents up to 20 % of the dry substance to be treated). The clear solution obtained after the decolorization by the activated carbon is further subjected to the sample separation at solid state and to the sample identification by HPLC and amino acid analyzer. D- Sample separation at solid state 1- Evaporate the elute solution in beaker at 60ºC - 70ºC under the atmospheric pressure. 2- Weigh the finally collected solid substances in beaker (X2 g) to estimate the percentage of the total amino acids separated at solid state to the dry matter of juice sample (W/W). E- Sample identification 1-HPLC of amino acids The apparatus used is Waters 600E Multisolvent Delivery System, Pico-Tag Analysis Column (column P mm, pre-column P 60 4 mm), Waters 484 Detector and Workstation with 815 Baseline Program (U.S.A). The analysis was carried out using a gradient of Pico-Tag solvent A, B at 40º C and flow rate 1 ml/min. detection of the separated PTC-amino acids (phenylthiocarbmyl) at 254nm wavelength. The buffer system used for separation is 50 mm sodium acetate ph 5.45 as buffer A and 70% acetonitrile 32 mm sodium phosphate ph 6.1 as buffer B. the program is run using a gradient of buffer A and buffer B with an initial 7% buffer B concentration and ending with 60% buffer B 6 TL2.1/

7 concentration at the end of the gradient. Before injection the sample, the instrument was calibrated by two injections of the standards (concentration 4 mmol/amino acid). 2-Amino acid analyzer The amino acids are separated on a cation exchange resin with buffers of carefully defined salt concentrations and ph. The resin, on which the ion exchange takes place, consists of small spherical beads of polystyrene, sulphonated to provide an electrical charge and reacted with divinyl-benzene to achieve the required degree of cross linkage between the polymerized chains of styrene. The sodium buffer system (three different ph values) was used for this particular analysis. Eluent from the ion exchange column is passed through a Teflon coil placed in boiling water bath. Before entering the coil the column effluent is mixed with an acetate buffer containing the reduced ninhydrin. This compound reacts with amino acids forming a dye complex. The absorption is measured at 440 and 570 nm in a flow photometer and registered on the chart of a recorder. Results and Discussion Satisfactory results obtained from this preliminary investigation of amino acids extraction from juice using ion exchange chromatography techniques as shown in Figs (1-7). RESEARCH NATIONAL CENTER, AMINO ACIDS PROJECT, AMINO ACID CHROMATOGRAPHY peak no. Conc. mg/100ml Glutamic Serine Isolucine Lucine Lysine Fig. 1: Separation of amino acids from beet juice without sample pretreatments (cold digestion). 7 TL2.1/

8 Fig. 2: Separation of free amino acids from beet juice by the pretreatment column filled with amberjet 1200 NA cation exchange resin during beet season of 2004/05. Fig. 3: Separation of proteinic amino acids from beet juice by the pretreatment column filled with Amberjet 1200 NA cation exchange resin during the beet season of 2004/05. * chromatographic peak areas were not be identified using a data analysis system and then checked with free and proteinic amino acids standard. 8 TL2.1/

9 Phosphoserine * Taurine* Phosphoethanolamine* Threonine* Serine* * Arginine* Fig. 4 Separation of free amino acids from beet juice by the pretreatment column filled with amberjet 1200 NA cation exchange resin during the beet season of 2006/07. Fig. 5: Separation of proteinic amino acids from beet juice by the pretreatment column filled with Amberjet 1200 NA cation exchange resin during the beet season of 2006/07. * chromatographic peak areas were not be identified using a data analysis system and then checked with free and proteinic amino acids standard. 9 TL2.1/

10 Fig. 6: Free amino acids standard, standard program P 2 on pre-column type H 60 4 mm. Fig. 7: Proteinic amino acids standard, standard program H 1 on pre-column type H 60 4 mm. 10 TL2.1/

11 Some general remarks could be made regarding the extraction of the amino acids from beet juice by using Amberjet 1200 NA cation exchange resin during the two successive beet seasons of 2004/05 and 2006/07. As shown in figure (1) it can observe that the way of preparing the final juice appeared to be important as well in the development of a successful sample pretreatment procedure. Due to the presence of small particulates or impurities/ colloids in the cold digest of juice samples, a reduction of the flow rate through the columns was observed and sometimes even a complete blockage of the column occurred and then the amino acids become less retarded and a poorer peak-resolution is obtained after a few days of analysis on the HPLC system. Moreover a difference between the percentage of total amino acids separated in sample and solid state using chromatography were observed. Only a warm aqueous digestion results in a final clear filtrate, which enables the subsequent sample treatment. That is why we decided to use a warm aqueous digestion. The colorants of juice were observed in the solution of sample, which seems to be more or less irreversible as turned out from visual inspection. That's why we decided to use a column of active carbon. The active carbon in the second-treatment picks up colorants as well from the samples. It might be interesting to study the maximum number of cycles till the active carbon becomes exhausted. Particularly the high concentration of sucrose in the filtrate will interfere in the chromatographic separation and detection of amino acids. Therefore, it was recommended for removing the excess of sugar prior to the chromatographic analysis in order to obtain a full-spectrum result of the amino acid composition. Sample preparation (solid phase extraction) is necessary to remove the surplus of sucrose, which otherwise demonstrates too much interference in the analysis. It can notice from Figs (2-3) that a total of fourteen amino acid, which were identified, could be extracted from beet juice. The greatest proportion of the amino acids extracted from beet juice consists of glutamic acid, phosphoserine, carnosine, and glycine being 1.45, 0.89, 0.57 and 0.42 % dry matter respectively, while the proportion of total amino acids extracted from beet juice sample being 5, 44% dry matter (W/W). These results was nearly similar to those reported by the authors Clarke and Pepperman [3], who's indicated that the effect of soil type on nutrient content illustrated that juices produced from beet grown on organic soils contained 5-6% amino acids as compared to 3% for juices from mineral soils. 11 TL2.1/

12 From the chromatograms in Figs (4-5) it can be concluded that there were a total of twenty different amino acid extracted from beet juice. It can be also noticed that the greatest proportion of the total amino acids extracted from beet juice consists of glutamic acid, alpha-amino adipic acid being 1.35 and 1.37% dry matter respectively. The results of analysis carried out on the amino acids, which extracted from juice by the pretreatment column during the two successive beet seasons under the same conditions of experiments, showed that the main amino acids of this particular juice were being glutamic acid, aspartic acid, threonine, glycine and phosphoserine. Moreover, there are some differences between the two analyses as shown in figs (4-5). The most striking difference between the two analyses is the presence of large quantities of alpha-amino-adipic acid, tryptophan and alanine in second analyses, which were absent or indiscernible in the first analysis. These results indicated that there are indeed some qualitative and quantitative differences of the amino acids extracted during the two successive beet season. It might be interesting to study the factors that caused these differences as N-fertilization, beet variety, soil type and climate conditions. Bruijn and Bout [4] indicated the variation in the amino acid composition of sugar beet and their absolute level of one variety of sugar beet, named Auris, which grown on different soil types was clay soil 21.7, sand soil 27.5 and high peat soil 27.9 mmol/kg beet. On the other hand, Baker [2] reported that the chemical composition of beet juice shows wide variations. The composition data reflect these variations were compiled from analysis presented in several publications, where the ranges of crude ash was , crude protein and crude fibre %. The protein quality of beet juice is rather high, as reported by Barrett [7], were indicated in the present study where a total of 10 essential amino acids were extracted from beet juice as shown in figs (4-5) glutamic acid, valine, methionine, leucine, cystine, phenylalanine, histidine, tryptophan, lysine and agrinine. The present results also identically to Edye report [8] which indicated that there were 10 essential and 13 nonessential for swine and 14 for poultry. However Wiggins [5.6] indicated in several publics that the following ten substances were present in beet: asparagines, aspartic acid, glutamic acid, serine, glycine, alanine, γ-amino butyric acid, lysine, valine and leucine or isomers. When the chromatograms of the standard amino acids, as shown in figs (6-7), are checked with the chromatograms of the juice sample, it becomes obvious that in the latter case there are a proteinic amino acids 12 TL2.1/

13 could be identified as threonine and tyrosine. However, there are a free amino acids could be identified as phosphoserine, taurine, phosphoethanolamine, threonine, serine and agrinine Calculation the quantities of matrix components collected at solid state The concentration of juice solution = 25 g /200 5 ml = The weight of residue (solid state) = X2- X1 = The percentage of residue (solid state) = / = 5.44 % dry matter Estimating the quantities of matrix components collected using a data analysis system Chromatographic peak areas are quantified using a data analysis system that attached to the amino acid analyzer. The results of this investigation are summarized in Tables 2 and 3. The quantities of total amino acids extracted from beet juice, which are identified in chromatogram, shown in Table 2 = µg/ml. Purified solution contains the amino acids which are 5 times diluted compared to the original extract from the warm juice digestion = = µg/ml = µg/5ml The percentage of total amino acid in juice sample = / = 4.29% dry matter. The difference between the percentage of total amino acids in sample and solid state due to the chromatographic peak areas which are not identified using a data analysis system. (unknown amino acid*). The total free nitrogen as animal feeding = = %. 13 TL2.1/

14 Table 2: The matrix components collected of the juice, by the pretreatment column filled with AMBERJET 1200 NA resins; average of measurements in (µg/ml juice). Name Concentration * µg/ml juice Concentration % Aspartic acid Threonine Serine Glutamic acid Glycine Valine Methionine Isoleucine Leucine Tyrosine Phosphoserine Phosphoethanolamine Cystine Carnosine Total amino acids % * Original concentrations multiplied by 5 Table 3 : The matrix components collected of the juice at another season of sugar beet, by the pretreatment column filled with AMBERJET 1200 NA resins; average of measurements in (µg/ml juice). Name Concentration * µg/ml juice Concentration % Aspartic acid Serine Glutamic acid Glycine Valine Methionine Leucine Tyrosine Cystine Carnosine α- Aminoadipic acid Alanine Beta-Alanine Phenylalanine Betaaminoisobutyric Gamaamino N butyric Histidine Tryptophan Lysine Arginine Total amino acids % The total free nitrogen * Original concentrations multiplied by 5 14 TL2.1/

15 References [1] R. A. Mcginnis, "Ion Exchange Nonsucrose Elimination." In: R.A. Mcginnis, (Third eds.), "Beet-Sugar Technology ", Beet Sugar Development Foundation, 1971, p [2] R.R. Baker, "The chemical composition of beet." Association of American Feed Control Officials, Official publication, AAFCO, C.R. Spooner, Department of Agriculture, Atlanta, 1998, pp [3] M.A. Clarke and A.B. Pepperman, "Contents of nitrogen-containing organic compounds and Amino acids in beet." Proceeding of the Sugar Processing Research Conference, pp (1998). [4] J.M. Bruijn and M.M. Bout, "HPAEC analysis of amino acids in sugar beet samples: Method development and application." Association AVH, 7 th Symposium, Reims, pp , marsh (2000). [5] B.K. Wiggins and R.S Williams, "Estimating of major amino acids constituents in beet juice or molasses." India Sugar Technologist's Association, pp (1998). [6] R.C.S. Partt and R.P. Wiggins, "Strip chromatography of beet juice." South African Sugar Technologist's Association; 43 (119), pp (2001). [7] G.C. Barrett, "Amino acids." In: J.S. Davies (eds.) "Amino Acids, Peptides, and Proteins." London, Royal Society of Chemistry, 1996, pp [8] L.A. Edye, "Nutrient of Domestic Animals." No. 2. "Nutrient Requirements of Swine", In: National Academy of Sciences/National Research Council. Washington, pp (1989). [9] A.A. Zerban, "Chemical separation of glutamine, asparagines and tyrosine from beet juice." India Sugar Technologist's Association, August, pp (2001). 15 TL2.1/