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Abstract
SUMMARY
The amino acids in molasses are scarcely studied and there are few contradictory reports dealing with the number and concentration of the amino acids therein. Amino acids are added to ration to increase the nutritional value. It is thought that amino acids can be separated from molasses using ion exchange chromatography techniques.
This study has been involved for years the application of ion exclusion chromatography to enhance the recovery of amino acids from beet molasses. Laboratory-scale tests of a simulated moving ion exchange bed chromatography system for amino acids recovery from sugar beet molasses were initiated in October 2004.
The technology developed in this study led to construction and start-up of the process-scale simulated moving ion exchange bed chromatography system for amino acids extraction from molasses.
Continuing research on ion exclusion applications in sugar beet molasses treatment has led to the licensing of other systems in Europe and the United States. Most recently, interest has been directed to the purification of diffusion juice directly by ion exclusion.
This preliminary investigation will give an idea of the applicability of ion exclusion to extract of amino acids from molasses to license this project in Egypt. The thesis includes three chapters:
I. The first chapter outline defining technical terms, providing a condensed description of the various stations in the beet sugar manufacture and lengthy in the composition and commercial use of blackstrap molasses.
Blackstrap molasses is the by-product (or end product) of beet and cane sugar manufacture or refining; it is the heavy, viscous liquid separated from the final low grade massecuite from which no further sugar can be crystallized by the usual methods. It is generally described as inedible because it is not used for human consumption. There are three defines categories of molasses: real molasses is the mother liquor from which sucrose crystals have been separated. Para molasses is obtained during the manufacture of other sugar (glucose, lactose, xylose). Pseudo molasses is what is produced directly, such as high-test molasses from raw cane and beet syrup.
As is often found with many industrial by-products, the chemical composition of molasses shows wide variation. Its composition is influenced by factors such as soil type, ambient temperature, moisture, season of production, variety, production practices at a particular processing plant, and by storage variables.
Consequently, considerable variation may be found in nutrient content, flavor, color, viscosity and total sugar content. The composition data reflect these differences since these figures were compiled from analysis presented in several publications, Crude ash 6.6 – 10.0, Crude protein 6.6 – 11.1, Crude fibre 0.0 – 0.3, Sucrose 43.0 – 50.5.
The levels of the amino acid are comparable to those reported earlier in the introduction, Curtin [19] indicated that the beet composition, consequently molasses, depends considerably on the growing conditions of the plants such as location, climate and agronomical factors, mainly fertilising, variety and population density.
Bruijn and Bout [39] developed a method for amino acid analysis 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 sugar beet, named Auris, which was grown on different soil types: clay soil 21.7, sand soil 27.5 and high peat soil 27.9 mmol/Kg beet.
Clarke and Pepperman [41] achieved that about 20 % of the total mass consists of non-sucrose organic matter, in particular of non-protein nitrogen (NPN) containing substances, such as betaine 4- 5 %. In addition molasses contains free and bound amino acids and pryrrolidone carboxylic acid (a conversion product of glutamine) 3- 4 % and amino acids sugar complexes 1-2 %.
The work of Partt and Wiggins [118] achieved that the first relatively complete identification of the amino acid in beet juice and showed that the following ten substances were present: asparagines, aspartic acid, glutamic acid, serine, glycine, alanine, γ-amino butyric acid, lysine, valine and leucine or isomers.
The molasses types contain significant levels of crude protein (N x 6.25). Also, the nitrogenous materials which are present consist mainly of non-protein nitrogen compounds which include amides, albuminoids, amino acids and other simple nitrogenous compounds. The effect of soil type on nutrient content is well illustrated that molasses produced from cane grown on organic soils contained 7-10% protein as compared to 3% for molasses from mineral soils.
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.
The term for the essential amino acid that is present in the lowest amount in the feed. Essential amino acids are required in the ration in definite proportions. Amino acids may supplement each other when two different protein feeds have different amounts of a limiting amino acid. Because of this supplementary effect, it is recommended that more than one source of protein be supplied in the diets of non-ruminants.
Also, illustrates the specific uses of the amino acids: Chelating / complexing agents for cation nutrients, plant growth regulators, substrate for microbiological products, and fertilizer source of nitrogen.
Most amino acids were produced by the hydrolysis of various protein sources. These may be plant, animal, or microorganism derived. A number of amino acids are produced by fermentation. The fermentation may take place on a culture media composed of grains, sugar, molasses, 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.
Chromatographic separation is used to separate valuable components from molasses. Typical products, which are currently in industrial scale production in addition to sugar, are betaine and inositol. The recovery of the other components can significantly improve the economy of sucrose recovery.
Chromatographic separation can also be applied to treat intermediate juices in the beet sugar industry. Low green or B- molasses separation is already in industrial use. Thick juice separation has also been suggested. The positioning of the chromatographic step in the beet sugar process has distinct effects on the recovery of sucrose and especially on the recovery of the other components. The resolution of the peaks changes due to changing composition of the feed material.
Recent development includes a patented two-stage process. It is a combination of two chromatographic fractionators to improve the recovery and the purity of overlapping components, like sucrose and betaine. It is uniquely suitable for recovering multiple value-added products from the process streams of the sugar industry.
A more detailed analysis of the group of α-amino nitrogen compounds in relation to, for instance, beet variety, fertilization, soil type and climate conditions might give valuable information whether (and how) it could be possible to further improve the internal quality of sugar beets.
Headquarter for productions of amino acids in the world were: Germany, France (Paris), USA, China (2001)
Production Plants for Pharmaceutical Grade L-amino acids
- Ham, France (Extraction from Proteins hydrolyzates),
- Konstanz, Germany (Enzymatic resolution),
- Nanning, China (Biocatalysis and fermentation 2001)
Production Plants for Feed Grade Aminoacids by Synthesis and Fermentation
Blair, Mobile , USA (L-lys ,DL-met)
Antwerp, Belgium (DL-met)
Wesseling, Germany (DL-met)
Slovakia (L-lys, L-thr)
Twenty different amino acids are present in protein hydrolysates, and other less common amino acids exist naturally and have biological functions. A growing number of non-protein amino acids that do not occur in nature have been synthesized. Except for glycine, all amino acids resulting from protein hydrolysis possess rotary optical activity. Amino acids that occur in plant or animal tissues are almost always in the L- enantiomer. This stereoisomerism is due to the presence of an asymmetric carbon atom.
II. The experimental part in chapter two involves the detailed description of the experiments carried out to develop the technology of extraction amino acids from molasses by ion exchange bed chromatography. The laboratory experiments were performed on the same sample of molasses during the two crushing seasons 2004/2005-2006/2007.
Many tests were represented different retention time and concentrations of molasses sample on column (from ½ to 1 hour), different type of resin (AG 50W-X8 cation exchange resin in the hydrogen form, 15 cm high × 3 cm dia and AMBERJET 1200 NA 74132 cation exchange resin, 15 cm high × 3 cm dia.), different seasons of beet crushing (2004/2005-2006/2007), different concentrations of buffering solutions (2-5%) and different pH (isoelectric point 2-6).
The separation process designated as an ion exclusion separation; in the course of this study examples of several different separation mechanisms encountered.
These include:
1- Ion exclusion separation which takes advantage of the fact that charged species (cations and anions) diffuse into the ionic matrix of ion exchange resin beads with more difficulty than small neutral molecules such as disaccharides and monosaccharides.
2- Size exclusion reflects the lower rate of diffusion of large molecules into the small pores of the resin matrix. Size exclusion allows the separation of neutral molecules, such as carbohydrates, of various sizes.
As might be expected, these effects can both operate in varying degrees depending on the size and ionic nature of the species under consideration.
The principles for the chromatographic separation of amino acids on a cation exchange resin are essentially based on:
- Acid-base properties, i.e. charge, of the amino acids;
- Relative polarity of (neutral) amino acids;
- Non-ionic, hyDROPhobic interactions with the styrene backbone of the resin.
The results in each experiment were therefore comparable with each other. The laboratory experiments were conducted to cover the following processes: Sample preparation and proteins hydrolysis, sample pretreatment, removal of neutral (and anionic) components in liquid samples, color removal, sample crystallization and HPLC of amino acids at the same conditions and the same period.
III. Chapter three illustrates the results and discussion of this investigation. Acid-base properties i.e. charge, of the amino acids; relative polarity of (neutral) amino acids and non-ionic, hyDROPhobic interactions with the styrene backbone of the resin, these phenomena in combination with the prevailing pH of the eluent can be proposed to explain the order of retention in a cation exchange separation of amino acids. Consequently, the physico-chemical properties of the applied cation exchange resin as well as the pH and ionic strength of the eluent (gradient) will determine the quality of the amino acids separation eventually achieved.
The way of preparing the final molasses appeared to be important as well in the development of a successful sample pretreatment procedure. Due to the presence of small particulates/ colloids in the (paper) filtered cold digest of molasses samples, a reduction of the flow rate through the columns was observed and sometimes even a complete blockage of the column occurred. Only a warm aqueous digestion results in a final clear filtrate, which enables the subsequent sample treatment.
The active carbon in the second-treatment pick up colorants as well from the samples, which seems to be more or less irreversible as turned out from visual inspection. That’s why we decided to discard a column after two cycles of usage. It might be interesting to study the maximum number of cycles till the resin becomes exhausted.
The ”A” column, which is particularly suitable for the separation of saccharides, demonstrates a fair separation of amino acids too; after optimisation of both the eluent composition and the applied gradient.
The technology developed in this work led to construction and start-up of the process-scale simulated moving ion exchange bed chromatography system for amino acids extraction from molasses. A good promising result was achieved such as:
1- Chromatographic peak areas are identified and quantitated using a data analysis system that is attached to the amino acid analyzer system. The percentage of total amino acid in molasses sample W/W = 5.44 % dry matter with a total of 20 different amino acids, which are identified, extracted from molassese, the proportion of amino acids extracted consists of aspartic acid, serine, glutamic acid, glycine, valine, methionine, leucine, tyrosine, cystine, carnosine, alpha-amino adipic acid, alanine, beta-alanine, phenylalanine, betaaminoisobutyric acid, gamaamino-N- butyric acid, histidine, tryptophan, lysine, arginine: 0.71%, 0.27%, 39.51%, 1.01%, 0.79%, 0.44%, 0.58%, 0.08%, 1.94%, 2.24% 41.51%, 0.67%, 1.00%, 1.43%, 1.91%, 2.13%, 0.25%, 2.21%, 0.75% and 0.57% of total amino acids respectively.
2- The difference between the percentage of dry substances and the percentage of total amino acid in sample using chromatogram due to the chromatographic peak areas which are not identified and quantitated using a data analysis system.
3- The total nitrogen free extract as animal feeding = 6.25 × 5.44
= 34 %.
4- The protein quality of beet molasses is rather high, because of a high proportion of essential amino acids extracted, 10 essential amino acids from a total 12 of essential amino aicds, (glutamic acid, valine, methionine, leucine, cystine, phenylalanine, histidine, tryptophan, lysine and agrinine).
5- The P column, which is particularly suitable for the separation of saccharides, demonstrates a fair separation of amino acids too; after optimisation of both the eluent composition and the applied gradient. Although the final solutions obtained after the pretreatment were free of sugars, the recovery of the amino acids appeared to be less than 100 %; range of about 40% Moreover, this phenomenon was not reproducible, particularly with respect to ”real” beet molasses extract samples (checked with standard addition). A possible explanation might be a too low exchange capacity of the resin AG 50W-X8 in the P columns.
6- In contrast to this, better results were achieved using the ”A” pretreatment column (filled with AMBERJET 1200 NA 74132 cation exchange resin, 15 cm high × 3 cm dia.); i.e. a reproducible recovery of around 100 %. The A column has been specially designed for the separation of amino acids. Indeed a better resolution of the separation of amino acids is obtained on this column.
7- It has been investigated the hydrolysis of proteins to their constituent amino acids from a combination of acid in the pretreatment of molasses. Glycine or any other amino acid can be separated and crystallized. This would be a natural method of amino acid production.
8- It has been also investigated if the amide glutamine is subject to any hydrolysis into glutamic acid and/or pyrrolidone carboxylic acid during the sample pretreatment. However, it could be ascertained that the glutamine, and so presumably asparagine, changed under the sample pretreatment conditions.
9- The way of preparing the final molasses appeared to be important as well in the development of a successful sample pretreatment procedure. Due to the presence of small particulates/ colloids in the (paper) filtered cold digest of molasses samples, a reduction of the flow rate through the columns was observed and sometimes even a complete blockage of the column occured. Only a warm aqueous digestion results in a final clear filtrate, which enables the subsequent sample treatment.
10- The active carbon in the second-treatment pick up colourants as well from the samples, which seems to be more or less irreversible as turned out from visual inspection. That’s why we decided to discard a column after two cycles of usage.
11- The benefit of the sample pretreatment is obvious. Normally, the mono-, di- and trisaccharides eluted in the isocratic part of the chromatogram, somewhere between 3 and 9 min. and, thus, would have interfered with the separation and detection of amino acids.
12- Due to fouling of the column by certain impurities present in the samples of molasses, the amino acids become less retarded and also a poorer peak-resolution is obtained after a few days of analysis on the HPAEC system. The retention of the amino acids can be restored by washing the column with 1 M HCl for about 1.5 hour at 0.2 ml/min. Before and after this washing the column has to be flushed with water in order to prevent any contact of the acid with ammonia.
13- Molasses must be fed into the column at 25 g/200 ml concentration. A higher concentration this tends to produce severe peak tailing due to the high viscosity.
14- 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.
15- The results showed that there are indeed considerable differences in the quantities and quantitative of amino acids present in molasses produced from different varieties examined. It also quite clearly indicated that the total amino acid content of the molasses falls as the beet nears maturity.