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Treatments of water hyacinth tissue to obtain useful products
Biological Wastes 33 (1990) 263-274
Treatments of Water Hyacinth Tissue to Obtain Useful Products Siegfried Bolenz, Helmy Omran & Karlheinz Gierschner Institut fiir Lebensmitteltechnologie, Universit~it Hohenheim, Garbenstrasse 25, D-7000 Stuttgart 70, FRG (Received 5 January 1990; revised version received 15 March 1990; accepted 23 March 1990)
ABSTRACT The water hyacinth (Eichhornia crassipes) could be an inexpensive source of protein for animal, and possibly human, nutrition. The fibre is usable for ruminants. This is a pilot study on processing the plant. The samples used came from Egypt. The tissue contains many air-filled intercellular spaces and soaks up water, but is also tough as a result offibres. It contains sharp needles formed by Ca-oxalate. Though the concentration is not high enough for toxicity, the needles could harm the digestive tract." Owing to the tissue's toughness much energy must be spent in chopping it, which results in many destroyed cells. Polyphenoloxidases are activated and the resulting quinones react with the protein, making it indigestible. So the native enzymes have to be inactivated before further processing. This could be done by adding low quantities of sulphite. Lactofermentation, a prerequisite for silage production, is possible, but only after adding sugar and suppressing mould growth. Many methods were tested in the attempt to make the protein soluble. The application of NaOH as a solvent, and also the combination of pectinase/cellulase showed the best results. It was possible to concentrate the soluble protein by means of ultrafiltration. Based on the results we propose a processing scheme.
INTRODUCTION Water hyacinth (WH) belongs to the genus Eichhornia, which was described first in 1824. It originates in the A m a z o n and forms, together with another 263 Biological Wastes 0269-7483/90/$03.50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
264
Siegfried Bolenz, Helmy Omran, Karlheinz Gierschner
eight genera, the monocotyledon family Pontederiaceae. These plants grow in and dominate their freshwater habitat owing to their high vegetative propagation rate and to the robustness of their seeds. Today more than 50 countries suffer from the effects caused by the fast growth of the plant which include obstruction of shipping routes and reservoirs; losses of water in irrigation systems by evaporation; high consumption of dissolved oxygen by spoiled plant material; nesting area for Anopheles and other harmful organisms (Philipp, 1981; Gopal, 1987). Because of the fast growth and high productivity farmers have attempted to use the plant for animal feeding. It was found that the biological value of its protein is as high as that of potatoes or clover (Gopal, 1987). Unfortunately, the crude plants were unsuitable as the sole source of nourishment. When fed WH exclusively animals have died (Philipp, 1981). To make them acceptable WH need to be dehydrated and processed in a suitable way. The aims of the research were: to discover the reasons for the low digestibility and even toxicity if only WH is fed; development of proper treatments to avoid reactions causing indigestibility and to eliminate toxic elements; extraction and concentration of protein. The results could then form the basis of a commercial processing scheme, although further research would be needed to assess its feasibility. METHODS
Sample collection WH were gathered from small channels in the Giza region near Cairo, Egypt. After washing and cutting off the roots, the plants were packed and transported by plane to Stuttgart (FRG). Most of the samples were stored at - 18°C. Some portions, which were destined for microscopic examination, were kept a short time at 4°C.
Microscopic and physical examination of the tissue A phase-contrast microscope with built-in camera was used. The composition of needle-like cell structures was analysed with an electron beam X-ray micro analyser (Heinrich, 1981) and a laser microprobe mass analyzer (LAMMA 500; Kaufmann et al., 1979).
Chemical methods Oxalate was measured enzymatically by the Boehringer method (Boehringer, 1987) after pH adjustment to 1.0 by addition of HCI to dissolve Ca-oxalate.
Processing water hyacinth
265
This was carried out with homogenized plant material and, after homogenization and centrifugation at 6000 g for 10 min, with sediment and juice. Activity of polyphenoloxidase (PPO) was measured by colour reaction of the quinones formed with o-phenylenediamine (Jankov, 1962). Reducing sugars were determined by the Luff-Schoorl method (Buckenhiiskes, 1984) and enzymatically (Boehringer, 1986). Protein was measured by a Kjeldahl method (no. 35 LMBG) and by the Bio-Rad-Protein-Assay (Bradford, 1976; Bio-Rad, 1986). Fibres were determined by the van-Soest detergency method (Ohlde & Becker, 1982), ash and dry matter according to the standard methods (no. 35 LMBG).
Buffering capacity and lactic acid fermentation The buffering capacity of plant material is defined as the capacity of the tissue to buffer its pH against the acid released during fermentation. So it monitors the sugar content necessary to reach a pH below 4.1, which is microbiologically safe (Buckenhiiskes, 1985). In spontaneous fermentation experiments the plants were chopped, with addition of 1% NaCI in order to avoid spoilage before fermentation started. Two different amounts of sucrose were added as well as sorbic acid to one batch. Samples were stored at room temperature and pH measured during one month.
Extraction of protein For screening different extraction methods, two similar standard procedures were used. In each experiment, 250 g of plant material were blanched (5 min in water at 90°C) to inactivate PPO. In this step the material soaked up some of the blanching water. To equalize this, more water was added later on while chopping the material. Including blanching water an addition of 25 or 100% water in relation to fresh weight was carried out. The material was chopped in a laboratory cutter, similar to those used in the meat industry, and extracted. The mash was pressed by means of a hand-operated, hydraulic laboratory press up to a constant final piston pressure of 300 x 105 Pa. The turbid juice was centrifuged 10 min at 1500g. After that, the clear juice was concentrated by ultrafiltration. Instead of blanching, the material was chopped after addition of 600 ppm sulphite and 25 or 100% of water, both in relation to fresh weight. The water addition was not only necessary for the comparability of the data; without it it was not possible to get a mash from choppingmthe resulting material had a dry structure like freshly-cut grass. All the following steps were carried out identically as above.
266
Siegfried Bolenz, Helmy Omran, Karlheinz Gierschner
Ultrafiltration A laboratory ultrafiltration unit was used. The juice circulates between membrane plates and pump (tangential flow). Pressure can be set up 5 x 10 s Pa, depending on pump speed. The cellulose membrane is permeable to molecules up to approximately 10 000 Daltons, but this is influenced by the pressure. By measuring mass and protein contents of juice, concentrate and permeate, concentration factors (concentrate/juice) and losses could be calculated.
RESULTS A N D DISCUSSION
Digestional problems caused by the structure of the WH-tissue The stalk tissue contains intercellular spaces filled with air (Fig. 1), which soak up water while the animals are digesting. They drink more and feel replete, though having little material of nutritional value in their rumens. Microscopic examination reveals sharp needles (Fig. 2). Investigation of the needles by electron beam X-ray analysis showed calcium to be the cation. Laser microprobe mass analyses spectra of the needles were very similar to a Ca-oxalate standard. Finally oxalate was measured enzymatically in homogenized plant material. After centrifugation 92% of the total amount was found in the solid matter. This supports the thesis that the needles are formed of Ca-oxalate. Compared with some vegetables the total concentration is not high (1.26 g/kg fresh weight) so toxicity is not the main problem. Of greater importance is that t h e needles could damage the digestive tract of animals fed with WH through their sharpness, if they were not dissolved by digestive acid. To avoid these problems, the tissue must be chopped to eliminate the included air and to negate its ability to absorb water. After pressing and centrifuging, the juice with the soluble components is separated from the solid matter, which contains the needles. The solid pomace could be washed with acid to eliminate the acid-soluble Ca-oxalate and then processed to a ruminant fodder.
Problems caused by polyphenol oxidases (PPO) Owing to its content of spiral-shaped fibres (see Fig. 1) the tissue is very tough. Much energy is required to chop it, which in turn destroys many cells. When PPO encounter their substrate, the quinones and polymers formed
Processing water hyacinth
267
Fig. 1. WH stalk tissue, containing air-filled intercellular spaces and spiral-shaped fibers (original magnification x 100).
Fig. 2.
Sharp Ca-oxalate needles released from WH tissue (original magnification x 2000).
Siegfried Bolenz, Helmy Omran, Karlkeinz Gierschner
268
react with the protein (condensation, agglomeration; Liener, 1980), making it indigestible and hindering its extraction. To avoid this reaction the native enzymes must be inactivated before chopping. Blanching as well as addition of sulphite (600 ppm/fresh weight) were tested. Both methods worked, but the latter gave better protein yields. Lactic acid fermentation as prerequisite o f silage production
All the following data were calculated in relation to fresh weight. WH showed a low buffering capacity of 0.4 g lactic acid/100g, so the calculated amount of sugar for successful fermentation would be between 0.4 and 0.8%. However, the content of fermentable sugar was 0.52%, so it was concluded that a supplement was necessary. In the fermentation experiments this was also proved. Without addition of sugar the pH was not lowered enough and the batch spoiled rapidly (Fig. 3, batch 1). Addition of 0-4% sugar (resulting in a total content of 0.92%) was enough to reach a pH below 4 (batch 2), but problems were caused by moulds. The pH rose again after several days and the sample spoiled. A reason could be the content of air in the tissue, which stimulates the growth of aerobic organisms. This did not happen in batch 3, which contained sorbic acid in order to suppress the moulds. This batch also showed that an addition of only 0.2% sugar is enough to reach a low pH.
3H Batch 1 -~-
Batch 2 Batch 3
6
3
0
I
i
I
i
i
,I
,5
10
15
20
25
30
35
Days of Fermentation Fig. 3. Spontaneous fermentationof WH: (1) + 1% NaCI;(2) 1% NaCI + 0-4% sucrose;(3)
1% NaCl + 0-2% Sucrose+ 0-1% sorbic acid.
Processing water hyacinth
269
Fine chopping to remove the air in the tissue and avoiding air contact during fermentation may solve the problem of spoilage by moulds without addition of fungicides.
Chemical analysis of WH dry matter The main components of the sample's dry matter (6.2%) were: ash, 15%; protein, 17%; sugar, 7 %; hemicellulose, 22%; cellulose, 31%; lignin, cutin, 7%. The tissue contains a large amount of fibres. This makes it advisable to use the residues after protein extraction for feeding ruminants. The presence of lignin contributes to the toughness of the tissue which was previously noted. The plants used for the experiments were between two and three months old. Using younger plants with less fibre could possibly ease the processing.
Extraction of protein Disintegration of the tissue was necessary to make the protein extractable. The first experiments in trying to obtain an enzymatic maceration and liquefaction showed that it was difficult to extract the protein completely. The reason may be the presence of lignin and cellulose and the whole NaOH-Addt-
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Fig. 4. Protein extracted from 250 g W H (fresh weight) in screening extraction methods. The hatched part of the bars indicates the losses in the ultrafiitration step. PPO was inactivated by means of blanching (odd bar numbers) or sulphite addition (even numbers). For further description of extraction see text.
270
Siegfried Bolenz, Helmy Omran, Karlheinz Gierschner
chemical structure of the cell walls in monocotyledons, which can be different from that found in dicotyledons (Darvill et al., 1980). Different methods of protein extraction were screened. Besides each method standard procedures were used (see Methods; Extraction of protein, Ultrafiltration). The protein contents of the resulting juices and ultrafiltration concentrates were measured by means of the Bio-Rad method (Bio-Rad, 1986). Figure 4 shows the results. The total bar indicates the protein content of the juice, the hatched part of it the protein loss in the following ultrafiltration, which was carried out at a pressure of 2 × 105 Pa. Each method was carried out twice, with PPO inactivated by means of blanching (odd bar numbers in Fig. 4) or sulphite addition (even bar numbers) The latter generally yielded more protein. The different methods can be ordered into groups. The cited numbers are the bar numbers in Fig. 4. --Mechanical methods: chopping and pressing without further treatment showed poor yields. In methods 1, 2 25% water was added, in 3, 4 100%. - - A d d i t i o n of 5% NaC1 (related to fresh weight) as protein solvent gave slightly better results. 5, 6 is with 25% water, 7, 8 with 100%. --pH-adjustment to 8.5 by means of NaOH and pressing after a reaction time of 1 h gave the best results of all. The losses by ultrafiltration were high in the first screening experiments, but this problem was solved later on by using a higher ultrafiltration pressure. Batches 9, 10 25% water addition, 11, 12 100%. - - T h e r m a l and combined methods. 100% water was added and the batches heated 10 min at 95°C. This gave a very poor result (13, 14). In 15, 16, 5% NaC1 was added before heating; in 17, 18 pH was set to 8.5 by means of NaOH. The results were better, but still worse than similar methods without heating. It was concluded that heat treatment is ineffective. --Enzymatic methods. The pH of the batches was set to 4.5 by means of HCI to give optimum conditions for the enzymes. Reaction time was 1 h at 4 0 ° C with stirring. First, 0"2% (related to fresh weight) of an industrial pectinase (Pectinex Ultra-SP, NOVO) was used. The results were poor with 25% water addition (19, 20) or with 100% (21, 22). A mixture of 0-1% of the pectinase with 0.1% of a cellulase (Celluclast, NOVO) with 100% added water was better (23, 24). In the last, somewhat complicated procedure, 0.1% pectinase was applied with 25 % of water. After pressing, the resulting pomace was mixed with 75 % water, the pH was set anew and 0.1% cellulase was added. After a second reaction time and pressing the juice was added to the first one. These batches (25, 26) gave the best results of the enzymatic methods.
Processing water hyacinth
271
The most promising experiments (batches 10 and 24, 26 in Fig. 4) were repeated with some changes. The absolute values of the protein determination had been very low in the last experiments, and thus the Kjeldahl method was used as well as the Bio-Rad method to measure the protein content. The differences were large (see Fig. 5). Though taking into consideration that Kjeldahl crude protein also includes non-protein-N, we concluded from the discrepancy that the affinity of Bio-Rad's colouring agent to WH protein must be very low. In these experiments the batches were bigger (1 kg), but the results were comparable with the previous ones. PPO was inactivated by means of sulphite addition. Extraction with NaOH and 25% water addition (bar 10 in Fig. 4) had resulted in big losses after ultrafiltration. So the UF pressure was now increased from 2 to 3 x 105 Pa, which resulted in a lower permeability of the membrane. The pH of the batch was now set at 9.0. In the repeated enzymatic method a mixture of pectinase, cellulase and a macerating enzyme (Ultrazym, NOVO) with 0.15% of each component was used. Apart from this, nothing in the procedure of batch 24 (see above and 600
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Fig. 5. Protein extracted from 250gWH (fresh weight) while developing the most promising extraction methods and comparing protein determination by means of the BioRad and the Kjeldahl methods. The hatched part of the bars indicates the losses in the ultrafiltration step.
272
Siegfried Bolenz, Helmy Omran, Kariheinz Gierschner TABLE 1 Protein Contents of Processed WH, % by Weight of Material per cent
Pellet NaOH extraction Enzymatic extraction
58 51
Sludge 20 29
Juice 22 20
Fig. 4) was changed. Batch 26 was not repeated because the procedure had been evaluated as too complicated for practical use. The results may be summarized as follows (see Fig. 5) The extraction with N a O H still showed the best protein yields, although the difference from the enzymatic method was not as great in the Kjeldahl results as it was when the samples were measured by means of the Bio-Rad assay. The losses in ultrafiltration of NaOH-treated WH now were much less than in the first experiment, measured by Bio-Rad. They remained on the same level as the enzymetreated batch. Measured by Kjeldahl, UF losses were high in both batches, but this result may have been influenced by non-protein-N. The crude protein of products resulting from the processing was investigated. The contents in pressing pellets and centrifuge sludges (see Methods) were measured by means of the Kjeldahl method and the samples were weighed. These data compared with the ones already obtained for raw material and juices before ultrafiltration gave the results in Table 1. Approximately half of the total protein content was still found in the press cakes. These could be used for ruminants. The centrifuge sludge also contained a large amount of protein. By means of a good centrifuging technique it should be possible to separate only Ca-oxalate needles, which are relatively large, and not the smaller protein particles. Then the turbid juice could be concentrated by means of tangential flow ultrafiltration, which should be possible and would minimize the protein losses.
Concentration of soluble protein by ultrafiltration In all the above experiments a concentration factor (protein content of concentrate/juice) of 4 was applied, but factors up to 28 were tried as well. Measured by the Bio-Rad assay, normally 75% to 90% of the protein in the juice was recovered. The indefiniteness o f results caused by different analytical methods (Bio-Rad and Kjeldahl)has already been mentioned. Concentration of protein by means of ultrafiltration should not cause major problems, once the extraction method is optimized and suitable membranes and processing parameters have been selected.
273
Processing water hyacinth I WH Raw Materiall i IIFlne ChoppingU ~1 IITissue ~
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~
/ Additives
Fig. 6. Flow sheet of a proposed WH processing route.
CONCLUSIONS Based on the results of this pilot study, we designed a flow sheet for W H processing, which could be used as a basis for further research (Fig. 6).
REFERENCES Bio-Rad Company (1986). Bio-Rad-Protein-Assay, Instruction Manual. Bio-Rad Laboratories, Munich. Boehringer Company (1986). o-Glucose/o-Fructose UV-Test. Anleitung zur TestKombination, Boehringer Mannheim GmbH. Boehringer Company (1987). OxalsLiure UV-Test. Anleitung zur TestKombination, Boehringer Mannheim GmbH. Bolenz, Siegfried (1988). Versuche zum Aufschlul3 des Wasserhyazinthengewebes zwecks Gewinnung wertvoller lnhaltsstoffe, insbesondere des Proteins. Diploma thesis, lnstitut fiJr Lebensmitteltechnologie, Universit~it Hohenheim. Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgramm quantities of protein utilizing the principle of protein-dye binding. Anal Biochem., 72, 248-54.
274
Siegfried Bolenz, Helmy Omran, Kariheinz Gierschner
Buckenhiiskes, H. (1984). Untersuchung der technologischen Rahmenbedingungen fiir ein neues Verfahren zur Herstellung von-Sauerkraut in Kleinbehiiltern. Dissertation, Universitiit Hohenheim. Buckenhiiskes, H. (1985). Bestimmung der Pufferkapazitiit. Alimenta, 24(4) 83-8. Darvill, A., McNeil, M., Albersheim, P. & Delmer, P. D. (1980). The primary cell wall of flowering plants. In The Biochemistry of Plants, VoL 1, ed. P. K. Stumpf & E. E. Conn. Academic Press, New York. Gopal, Brij, (1987). Water Hyacinth, Aquatic Plant Studies 1. Elsevier Science Publishers BV, Amsterdam. Heinrich, K. F. J. (1981). Electron Beam X-ray Microanalysis. Van Nostrand Reinhold Company, New York. Jankov, S. J. (1962). Hitzeinaktivierung von Polyphenoloxidasen in einigen Fruchtsiiften. Fruchtsaft-Industrie, 7, 13-32. Kaufmann, R., Hillenkamp, F., Wechsung, R., Heinen, H. J. & Schiirmann, M. (1979). Laser Microprobe Mass Analysis: Achievements and Aspects. Scanning Electron Microsc., II, 279-90. Liener, I. E. (1980). Toxic Constituents of Food Plantstuffs (2nd edn). Academic Press, New York. Ohlde, G. & Becker, K. (1982). Suitability of cell wall constituents as predictors of organic matter digestibility in some tropical and subtropical by-products. Anim. Feed Sci. & Technol., 7, 191-9. Philipp, Ottmar (1981). Zur Verwertung der Wasserhyazinthe (Eichhornia crassipes (Mart.) Solms). Dissertation, Universitiit Hohenheim. No. 35 LMBG: Amtliche Sammlung der Untersuchungsverfahren fiir Lebensmittel, No. 35. Lebensmittel- und Bedarfsgegenstiindegesetz, Bundesrepublik Deutschland.
Treatments of Water Hyacinth Tissue to Obtain Useful Products Siegfried Bolenz, Helmy Omran & Karlheinz Gierschner Institut fiir Lebensmitteltechnologie, Universit~it Hohenheim, Garbenstrasse 25, D-7000 Stuttgart 70, FRG (Received 5 January 1990; revised version received 15 March 1990; accepted 23 March 1990)
ABSTRACT The water hyacinth (Eichhornia crassipes) could be an inexpensive source of protein for animal, and possibly human, nutrition. The fibre is usable for ruminants. This is a pilot study on processing the plant. The samples used came from Egypt. The tissue contains many air-filled intercellular spaces and soaks up water, but is also tough as a result offibres. It contains sharp needles formed by Ca-oxalate. Though the concentration is not high enough for toxicity, the needles could harm the digestive tract." Owing to the tissue's toughness much energy must be spent in chopping it, which results in many destroyed cells. Polyphenoloxidases are activated and the resulting quinones react with the protein, making it indigestible. So the native enzymes have to be inactivated before further processing. This could be done by adding low quantities of sulphite. Lactofermentation, a prerequisite for silage production, is possible, but only after adding sugar and suppressing mould growth. Many methods were tested in the attempt to make the protein soluble. The application of NaOH as a solvent, and also the combination of pectinase/cellulase showed the best results. It was possible to concentrate the soluble protein by means of ultrafiltration. Based on the results we propose a processing scheme.
INTRODUCTION Water hyacinth (WH) belongs to the genus Eichhornia, which was described first in 1824. It originates in the A m a z o n and forms, together with another 263 Biological Wastes 0269-7483/90/$03.50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
264
Siegfried Bolenz, Helmy Omran, Karlheinz Gierschner
eight genera, the monocotyledon family Pontederiaceae. These plants grow in and dominate their freshwater habitat owing to their high vegetative propagation rate and to the robustness of their seeds. Today more than 50 countries suffer from the effects caused by the fast growth of the plant which include obstruction of shipping routes and reservoirs; losses of water in irrigation systems by evaporation; high consumption of dissolved oxygen by spoiled plant material; nesting area for Anopheles and other harmful organisms (Philipp, 1981; Gopal, 1987). Because of the fast growth and high productivity farmers have attempted to use the plant for animal feeding. It was found that the biological value of its protein is as high as that of potatoes or clover (Gopal, 1987). Unfortunately, the crude plants were unsuitable as the sole source of nourishment. When fed WH exclusively animals have died (Philipp, 1981). To make them acceptable WH need to be dehydrated and processed in a suitable way. The aims of the research were: to discover the reasons for the low digestibility and even toxicity if only WH is fed; development of proper treatments to avoid reactions causing indigestibility and to eliminate toxic elements; extraction and concentration of protein. The results could then form the basis of a commercial processing scheme, although further research would be needed to assess its feasibility. METHODS
Sample collection WH were gathered from small channels in the Giza region near Cairo, Egypt. After washing and cutting off the roots, the plants were packed and transported by plane to Stuttgart (FRG). Most of the samples were stored at - 18°C. Some portions, which were destined for microscopic examination, were kept a short time at 4°C.
Microscopic and physical examination of the tissue A phase-contrast microscope with built-in camera was used. The composition of needle-like cell structures was analysed with an electron beam X-ray micro analyser (Heinrich, 1981) and a laser microprobe mass analyzer (LAMMA 500; Kaufmann et al., 1979).
Chemical methods Oxalate was measured enzymatically by the Boehringer method (Boehringer, 1987) after pH adjustment to 1.0 by addition of HCI to dissolve Ca-oxalate.
Processing water hyacinth
265
This was carried out with homogenized plant material and, after homogenization and centrifugation at 6000 g for 10 min, with sediment and juice. Activity of polyphenoloxidase (PPO) was measured by colour reaction of the quinones formed with o-phenylenediamine (Jankov, 1962). Reducing sugars were determined by the Luff-Schoorl method (Buckenhiiskes, 1984) and enzymatically (Boehringer, 1986). Protein was measured by a Kjeldahl method (no. 35 LMBG) and by the Bio-Rad-Protein-Assay (Bradford, 1976; Bio-Rad, 1986). Fibres were determined by the van-Soest detergency method (Ohlde & Becker, 1982), ash and dry matter according to the standard methods (no. 35 LMBG).
Buffering capacity and lactic acid fermentation The buffering capacity of plant material is defined as the capacity of the tissue to buffer its pH against the acid released during fermentation. So it monitors the sugar content necessary to reach a pH below 4.1, which is microbiologically safe (Buckenhiiskes, 1985). In spontaneous fermentation experiments the plants were chopped, with addition of 1% NaCI in order to avoid spoilage before fermentation started. Two different amounts of sucrose were added as well as sorbic acid to one batch. Samples were stored at room temperature and pH measured during one month.
Extraction of protein For screening different extraction methods, two similar standard procedures were used. In each experiment, 250 g of plant material were blanched (5 min in water at 90°C) to inactivate PPO. In this step the material soaked up some of the blanching water. To equalize this, more water was added later on while chopping the material. Including blanching water an addition of 25 or 100% water in relation to fresh weight was carried out. The material was chopped in a laboratory cutter, similar to those used in the meat industry, and extracted. The mash was pressed by means of a hand-operated, hydraulic laboratory press up to a constant final piston pressure of 300 x 105 Pa. The turbid juice was centrifuged 10 min at 1500g. After that, the clear juice was concentrated by ultrafiltration. Instead of blanching, the material was chopped after addition of 600 ppm sulphite and 25 or 100% of water, both in relation to fresh weight. The water addition was not only necessary for the comparability of the data; without it it was not possible to get a mash from choppingmthe resulting material had a dry structure like freshly-cut grass. All the following steps were carried out identically as above.
266
Siegfried Bolenz, Helmy Omran, Karlheinz Gierschner
Ultrafiltration A laboratory ultrafiltration unit was used. The juice circulates between membrane plates and pump (tangential flow). Pressure can be set up 5 x 10 s Pa, depending on pump speed. The cellulose membrane is permeable to molecules up to approximately 10 000 Daltons, but this is influenced by the pressure. By measuring mass and protein contents of juice, concentrate and permeate, concentration factors (concentrate/juice) and losses could be calculated.
RESULTS A N D DISCUSSION
Digestional problems caused by the structure of the WH-tissue The stalk tissue contains intercellular spaces filled with air (Fig. 1), which soak up water while the animals are digesting. They drink more and feel replete, though having little material of nutritional value in their rumens. Microscopic examination reveals sharp needles (Fig. 2). Investigation of the needles by electron beam X-ray analysis showed calcium to be the cation. Laser microprobe mass analyses spectra of the needles were very similar to a Ca-oxalate standard. Finally oxalate was measured enzymatically in homogenized plant material. After centrifugation 92% of the total amount was found in the solid matter. This supports the thesis that the needles are formed of Ca-oxalate. Compared with some vegetables the total concentration is not high (1.26 g/kg fresh weight) so toxicity is not the main problem. Of greater importance is that t h e needles could damage the digestive tract of animals fed with WH through their sharpness, if they were not dissolved by digestive acid. To avoid these problems, the tissue must be chopped to eliminate the included air and to negate its ability to absorb water. After pressing and centrifuging, the juice with the soluble components is separated from the solid matter, which contains the needles. The solid pomace could be washed with acid to eliminate the acid-soluble Ca-oxalate and then processed to a ruminant fodder.
Problems caused by polyphenol oxidases (PPO) Owing to its content of spiral-shaped fibres (see Fig. 1) the tissue is very tough. Much energy is required to chop it, which in turn destroys many cells. When PPO encounter their substrate, the quinones and polymers formed
Processing water hyacinth
267
Fig. 1. WH stalk tissue, containing air-filled intercellular spaces and spiral-shaped fibers (original magnification x 100).
Fig. 2.
Sharp Ca-oxalate needles released from WH tissue (original magnification x 2000).
Siegfried Bolenz, Helmy Omran, Karlkeinz Gierschner
268
react with the protein (condensation, agglomeration; Liener, 1980), making it indigestible and hindering its extraction. To avoid this reaction the native enzymes must be inactivated before chopping. Blanching as well as addition of sulphite (600 ppm/fresh weight) were tested. Both methods worked, but the latter gave better protein yields. Lactic acid fermentation as prerequisite o f silage production
All the following data were calculated in relation to fresh weight. WH showed a low buffering capacity of 0.4 g lactic acid/100g, so the calculated amount of sugar for successful fermentation would be between 0.4 and 0.8%. However, the content of fermentable sugar was 0.52%, so it was concluded that a supplement was necessary. In the fermentation experiments this was also proved. Without addition of sugar the pH was not lowered enough and the batch spoiled rapidly (Fig. 3, batch 1). Addition of 0-4% sugar (resulting in a total content of 0.92%) was enough to reach a pH below 4 (batch 2), but problems were caused by moulds. The pH rose again after several days and the sample spoiled. A reason could be the content of air in the tissue, which stimulates the growth of aerobic organisms. This did not happen in batch 3, which contained sorbic acid in order to suppress the moulds. This batch also showed that an addition of only 0.2% sugar is enough to reach a low pH.
3H Batch 1 -~-
Batch 2 Batch 3
6
3
0
I
i
I
i
i
,I
,5
10
15
20
25
30
35
Days of Fermentation Fig. 3. Spontaneous fermentationof WH: (1) + 1% NaCI;(2) 1% NaCI + 0-4% sucrose;(3)
1% NaCl + 0-2% Sucrose+ 0-1% sorbic acid.
Processing water hyacinth
269
Fine chopping to remove the air in the tissue and avoiding air contact during fermentation may solve the problem of spoilage by moulds without addition of fungicides.
Chemical analysis of WH dry matter The main components of the sample's dry matter (6.2%) were: ash, 15%; protein, 17%; sugar, 7 %; hemicellulose, 22%; cellulose, 31%; lignin, cutin, 7%. The tissue contains a large amount of fibres. This makes it advisable to use the residues after protein extraction for feeding ruminants. The presence of lignin contributes to the toughness of the tissue which was previously noted. The plants used for the experiments were between two and three months old. Using younger plants with less fibre could possibly ease the processing.
Extraction of protein Disintegration of the tissue was necessary to make the protein extractable. The first experiments in trying to obtain an enzymatic maceration and liquefaction showed that it was difficult to extract the protein completely. The reason may be the presence of lignin and cellulose and the whole NaOH-Addt-
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Fig. 4. Protein extracted from 250 g W H (fresh weight) in screening extraction methods. The hatched part of the bars indicates the losses in the ultrafiitration step. PPO was inactivated by means of blanching (odd bar numbers) or sulphite addition (even numbers). For further description of extraction see text.
270
Siegfried Bolenz, Helmy Omran, Karlheinz Gierschner
chemical structure of the cell walls in monocotyledons, which can be different from that found in dicotyledons (Darvill et al., 1980). Different methods of protein extraction were screened. Besides each method standard procedures were used (see Methods; Extraction of protein, Ultrafiltration). The protein contents of the resulting juices and ultrafiltration concentrates were measured by means of the Bio-Rad method (Bio-Rad, 1986). Figure 4 shows the results. The total bar indicates the protein content of the juice, the hatched part of it the protein loss in the following ultrafiltration, which was carried out at a pressure of 2 × 105 Pa. Each method was carried out twice, with PPO inactivated by means of blanching (odd bar numbers in Fig. 4) or sulphite addition (even bar numbers) The latter generally yielded more protein. The different methods can be ordered into groups. The cited numbers are the bar numbers in Fig. 4. --Mechanical methods: chopping and pressing without further treatment showed poor yields. In methods 1, 2 25% water was added, in 3, 4 100%. - - A d d i t i o n of 5% NaC1 (related to fresh weight) as protein solvent gave slightly better results. 5, 6 is with 25% water, 7, 8 with 100%. --pH-adjustment to 8.5 by means of NaOH and pressing after a reaction time of 1 h gave the best results of all. The losses by ultrafiltration were high in the first screening experiments, but this problem was solved later on by using a higher ultrafiltration pressure. Batches 9, 10 25% water addition, 11, 12 100%. - - T h e r m a l and combined methods. 100% water was added and the batches heated 10 min at 95°C. This gave a very poor result (13, 14). In 15, 16, 5% NaC1 was added before heating; in 17, 18 pH was set to 8.5 by means of NaOH. The results were better, but still worse than similar methods without heating. It was concluded that heat treatment is ineffective. --Enzymatic methods. The pH of the batches was set to 4.5 by means of HCI to give optimum conditions for the enzymes. Reaction time was 1 h at 4 0 ° C with stirring. First, 0"2% (related to fresh weight) of an industrial pectinase (Pectinex Ultra-SP, NOVO) was used. The results were poor with 25% water addition (19, 20) or with 100% (21, 22). A mixture of 0-1% of the pectinase with 0.1% of a cellulase (Celluclast, NOVO) with 100% added water was better (23, 24). In the last, somewhat complicated procedure, 0.1% pectinase was applied with 25 % of water. After pressing, the resulting pomace was mixed with 75 % water, the pH was set anew and 0.1% cellulase was added. After a second reaction time and pressing the juice was added to the first one. These batches (25, 26) gave the best results of the enzymatic methods.
Processing water hyacinth
271
The most promising experiments (batches 10 and 24, 26 in Fig. 4) were repeated with some changes. The absolute values of the protein determination had been very low in the last experiments, and thus the Kjeldahl method was used as well as the Bio-Rad method to measure the protein content. The differences were large (see Fig. 5). Though taking into consideration that Kjeldahl crude protein also includes non-protein-N, we concluded from the discrepancy that the affinity of Bio-Rad's colouring agent to WH protein must be very low. In these experiments the batches were bigger (1 kg), but the results were comparable with the previous ones. PPO was inactivated by means of sulphite addition. Extraction with NaOH and 25% water addition (bar 10 in Fig. 4) had resulted in big losses after ultrafiltration. So the UF pressure was now increased from 2 to 3 x 105 Pa, which resulted in a lower permeability of the membrane. The pH of the batch was now set at 9.0. In the repeated enzymatic method a mixture of pectinase, cellulase and a macerating enzyme (Ultrazym, NOVO) with 0.15% of each component was used. Apart from this, nothing in the procedure of batch 24 (see above and 600
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Fig. 5. Protein extracted from 250gWH (fresh weight) while developing the most promising extraction methods and comparing protein determination by means of the BioRad and the Kjeldahl methods. The hatched part of the bars indicates the losses in the ultrafiltration step.
272
Siegfried Bolenz, Helmy Omran, Kariheinz Gierschner TABLE 1 Protein Contents of Processed WH, % by Weight of Material per cent
Pellet NaOH extraction Enzymatic extraction
58 51
Sludge 20 29
Juice 22 20
Fig. 4) was changed. Batch 26 was not repeated because the procedure had been evaluated as too complicated for practical use. The results may be summarized as follows (see Fig. 5) The extraction with N a O H still showed the best protein yields, although the difference from the enzymatic method was not as great in the Kjeldahl results as it was when the samples were measured by means of the Bio-Rad assay. The losses in ultrafiltration of NaOH-treated WH now were much less than in the first experiment, measured by Bio-Rad. They remained on the same level as the enzymetreated batch. Measured by Kjeldahl, UF losses were high in both batches, but this result may have been influenced by non-protein-N. The crude protein of products resulting from the processing was investigated. The contents in pressing pellets and centrifuge sludges (see Methods) were measured by means of the Kjeldahl method and the samples were weighed. These data compared with the ones already obtained for raw material and juices before ultrafiltration gave the results in Table 1. Approximately half of the total protein content was still found in the press cakes. These could be used for ruminants. The centrifuge sludge also contained a large amount of protein. By means of a good centrifuging technique it should be possible to separate only Ca-oxalate needles, which are relatively large, and not the smaller protein particles. Then the turbid juice could be concentrated by means of tangential flow ultrafiltration, which should be possible and would minimize the protein losses.
Concentration of soluble protein by ultrafiltration In all the above experiments a concentration factor (protein content of concentrate/juice) of 4 was applied, but factors up to 28 were tried as well. Measured by the Bio-Rad assay, normally 75% to 90% of the protein in the juice was recovered. The indefiniteness o f results caused by different analytical methods (Bio-Rad and Kjeldahl)has already been mentioned. Concentration of protein by means of ultrafiltration should not cause major problems, once the extraction method is optimized and suitable membranes and processing parameters have been selected.
273
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Fig. 6. Flow sheet of a proposed WH processing route.
CONCLUSIONS Based on the results of this pilot study, we designed a flow sheet for W H processing, which could be used as a basis for further research (Fig. 6).
REFERENCES Bio-Rad Company (1986). Bio-Rad-Protein-Assay, Instruction Manual. Bio-Rad Laboratories, Munich. Boehringer Company (1986). o-Glucose/o-Fructose UV-Test. Anleitung zur TestKombination, Boehringer Mannheim GmbH. Boehringer Company (1987). OxalsLiure UV-Test. Anleitung zur TestKombination, Boehringer Mannheim GmbH. Bolenz, Siegfried (1988). Versuche zum Aufschlul3 des Wasserhyazinthengewebes zwecks Gewinnung wertvoller lnhaltsstoffe, insbesondere des Proteins. Diploma thesis, lnstitut fiJr Lebensmitteltechnologie, Universit~it Hohenheim. Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgramm quantities of protein utilizing the principle of protein-dye binding. Anal Biochem., 72, 248-54.
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Buckenhiiskes, H. (1984). Untersuchung der technologischen Rahmenbedingungen fiir ein neues Verfahren zur Herstellung von-Sauerkraut in Kleinbehiiltern. Dissertation, Universitiit Hohenheim. Buckenhiiskes, H. (1985). Bestimmung der Pufferkapazitiit. Alimenta, 24(4) 83-8. Darvill, A., McNeil, M., Albersheim, P. & Delmer, P. D. (1980). The primary cell wall of flowering plants. In The Biochemistry of Plants, VoL 1, ed. P. K. Stumpf & E. E. Conn. Academic Press, New York. Gopal, Brij, (1987). Water Hyacinth, Aquatic Plant Studies 1. Elsevier Science Publishers BV, Amsterdam. Heinrich, K. F. J. (1981). Electron Beam X-ray Microanalysis. Van Nostrand Reinhold Company, New York. Jankov, S. J. (1962). Hitzeinaktivierung von Polyphenoloxidasen in einigen Fruchtsiiften. Fruchtsaft-Industrie, 7, 13-32. Kaufmann, R., Hillenkamp, F., Wechsung, R., Heinen, H. J. & Schiirmann, M. (1979). Laser Microprobe Mass Analysis: Achievements and Aspects. Scanning Electron Microsc., II, 279-90. Liener, I. E. (1980). Toxic Constituents of Food Plantstuffs (2nd edn). Academic Press, New York. Ohlde, G. & Becker, K. (1982). Suitability of cell wall constituents as predictors of organic matter digestibility in some tropical and subtropical by-products. Anim. Feed Sci. & Technol., 7, 191-9. Philipp, Ottmar (1981). Zur Verwertung der Wasserhyazinthe (Eichhornia crassipes (Mart.) Solms). Dissertation, Universitiit Hohenheim. No. 35 LMBG: Amtliche Sammlung der Untersuchungsverfahren fiir Lebensmittel, No. 35. Lebensmittel- und Bedarfsgegenstiindegesetz, Bundesrepublik Deutschland.