Biodegradable plastic made from bean products

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1 Industrial Crops and Products 11 (2000) Biodegradable plastic made from bean products E.M. Salmoral a, *, M.E. Gonzalez b, M.P. Mariscal a a Department of Chemistry, Engineering Faculty, Buenos Aires Uni ersity, Paseo Colon 850 (C.P. 1063), Buenos Aires, Argentina b Technological and Agricultural Unit, Ezeiza Atomic Center, National Atomic Energy Commission, Argentina Accepted 8 October 1999 Abstract The aim of the present work was to explore the suitability of two kinds of bean with typical protein quality and different starch structure to produce moulded specimens by compression. In this work we analysed the possibility of forming new plastic materials from two kinds of bean preparations; with different protein content, in the presence or absence of inherent cellulose material and other biopolymer such as starch. Significant differences were determined in the fine structure of glucopolysaccharides isolated from the different types of bean. Complete moulding of the specimens took place at 20 MPa at a temperature not higher than 140 C, for 7 min. The mechanical properties of the moulded plastics based on soluble protein black bean (BP) were superior in elongation, tensile strength and low water absorption than the soluble protein white bean (WP) variety. However, the addition of boric acid 3% (w/w, in total batch basis) to white bean plastic materials was effective in improving slightly the mechanical properties in the case of their whole flours products (WWF). The gamma radiation was effective to reduce water absorption only in plastics materials with cellulose starch content (BWF, WWF) and improved their mechanical properties. The gelatinization process of the moulded material was influenced by the predominant lineal structure of the carbohydrate content Elsevier Science B.V. All rights reserved. Keywords: Biodegradable plastics; Agricultural plastics; Bean protein; Gamma-irradiation 1. Introduction The use of renewable natural polymers in the manufacture of plastics grows in impulse and importance in the last years of the 20th century. Many major chemical companies are becoming increasingly interested in developing technologies * Corresponding author. Tel.: ; fax: addresses: esalmor@fi.uba.ar (E.M. Salmoral), maegonz@cnea.gov.ar (M.E. Gonzalez) for the manufacturing of products from crops. Among the products derived from the leguminous studied for manufacturing plastics, are the soy isolate prepared by precipitation at ph 4.5, with 90% protein; the soy concentrate, prepared by eluting soluble components from defatted soy flour with 70% protein and about 18% carbohydrate and soy flour with lower protein content, from 50 to about 34% (Kinsella 1979; Salt et al., 1982). In the present work we employed as raw material two leguminous varieties like Phaseolus ul /00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S (99)

2 218 E.M. Salmoral et al. / Industrial Crops and Products 11 (2000) garis L., from which there is evidence about their prehistoric existence in South America. Their greatest production is in the North West of Argentina. The black bean used is a cultivar noticeable for its high resistance to viral diseases of warm areas and it is eventually of high yield (Vizgarra, 1995). We have utilised the white bean cultivar because it is commercially important and has also been submitted to phyto-improvement by agronomic experiments. The seeds, which are typical of Argentina, were defatted; their polysaccharides, proteins and fiber isolated, purified and quantified. Starch is a mixture of two polysaccharides: amylose (in which predominates linear -1,4 glucan), and amylopectin (highly branched -1,6 glucan with branching points occurring through -1,6 linkages). The two components: amylose and amylopectin are present in varied ratios, depending on the vegetal source (Salmoral et al., 1993). In our case, the starch was used in addition to glycerol as plasticizers and was selected in order not to reduce the biodegradability of biopolymers. Boric acid is reported to be able to react with glycerol and starch forming a network among starch chains and glycerol molecules. It has been suggested the existence of crosslinks among starch, glycerol and boric acid. Crosslinks between OH- of the components caused by boric acid would enlarge the molecules and so prevent the segments for easy moving (Yu et al., 1998). In the present work, the addition of boric acid to the blend in a dose non toxic was studied. We examined the effect of gamma radiation on blends of proteins and defatted whole flour with respect to the mechanical properties and the water absorption of the molded plastics. Gamma radiation has been reported to be useful in the crosslinking of proteins (Ressouany et al., 1998), which would mean an increase in the tensile strength of the product. On the other hand, ionizing radiation acts on polysaccharides by breaking C C bonds and forming acid and reductive groups (Pruzinek and Hola, 1987) The objective was to compare two different varieties of crop products of legumes of Phaseolus ulgaris L. 2. Materials and methods 2.1. Materials Culti ation Phaseolus ulgaris L., black bean Tuc 500 and white bean Alule 91 were obtained from the Experimental Station of the National Institute of Agricultural Technology (INTA), from Tucumán and Salta, Argentina Soluble protein preparation Black bean soluble protein (BP) and white bean soluble protein (WP) (patent pending) were prepared in the laboratory using pulverized seeds; ground in a mill and defatted by the Soxhlet method. After treatment with 0.001M NaHSO 3 and the homogenization at ph not higher than 6. The material was filtered to separate the cellulose material. The supernatant was decanted and the rest of the suspension was concentrated by evaporation and finally lyophillized. It was kept at 4 C, and the protein content determined (50%) Whole flours Black bean whole flour (BWF) and white bean whole flour (WWF) were prepared in the laboratory by crushing the seeds, defatting the powder by the Soxhlet method and pulverizing the material with help of a mill until it could be sieved through a 230 ASTM mesh. It was kept at 4 C and the protein content determined (BWF; 32%, WWF; 30%) Glucopolysaccharides In the process of isolation the pulverized seeds were soaked in M NaHSO 3, homogenized and squeezed in cheesecloth thus obtaining a supernatant that was decanted. The precipitated was submitted to several washes with: NaCl in aqueous solution, NaCl in aqueous solution/ toluene (140:1), ethanol/water (1:3), (2:3) and (3:1) successively. The starch obtained was finally

3 E.M. Salmoral et al. / Industrial Crops and Products 11 (2000) washed, dried and defatted by the Soxhlet method. The purification process: the 1,4 1,6 glucopolysaccharides were purified in Biogel P6 ( mesh) before analysing its structural characteristics (the wavelength of maximum absorption and the polysaccharide spectra were analysed in a UV-VIS Beckman spectrophotometer). The structural analysis of the 1,4-1,6 linked glucopolysaccharides, was described in a previous paper (Tolmasky and Krisman, 1987; Salmoral et al., 1993). The proteins were quantified by Kjeldhal method and in the washing solutions by the Lowry method ( Lowry et al., 1951) Preparation and plasticization of mixtures Blend The starch (11.7%) of total weight gelatinized with water and the glycerol (21.7%) of total weight were incorporated to either the protein soluble fraction or the whole flour material. The water content of these blends was 28%. All proportions are w/w in total batch basis. The blends were kept at 4 C for 24 h in closed containers for stabilisation previously to the compression process Treatment with boric acid The blend was prepared with the addition of boric acid 3% (w/w in total batch basis) Specimen preparation Specimens were prepared from bean products (soluble protein preparation and whole flour preparation) following the same technique employed for soybean products described elsewhere (Salmoral et al., 1998). The carefully homogenised blends were left to stand to reach a temperature of 23 C in closed containers and were immediately submitted to compression-moulding under the following conditions: compression load, 20 MPa; molding temperature, C and molding time 7 min. Plates 0.7 mm thick were obtained. Specimens for microtensile tests according to ASTM D-1708 (ASTM 1984) were cut out of the plates and conditioned at 23 C, 70% relative humidity for 48 h. The borders and edges were polished as well as the surfaces with abrasive sandpaper. This kind of specimens was chosen in order to be able to prepare large numbers of specimens with relatively small amounts of material, for each kind of composition tested. The mechanical properties were measured in this work only for comparative purposes among the different materials employed, not intending to define basic properties of the materials Irradiation Irradiation of some of the blends was carried out with gamma beams from a Co-60 source, at a dose rate of 9.5 kgy/h to a total dose of 50 kgy. The samples were packed in polyethylene flasks in the presence of air. The temperature in the irradiation chamber never surpassed 30 C. The dosimetry was performed by means of dichromate dosimeters for high doses. The homogeneity of dose within the samples was of 30%. Irradiation was always performed after 24 h of blend preparation. The time from irradiation to compression moulding was also of about 24 h. In all cases irradiated and non-irradiated samples were processed and tested in the same batch Water absorption Water absorption was measured according to ASTM D (ASTM, 1995). Samples 8.0 mm 15 mm and 1.5mm thick were conditioned in an oven for 24 h at 50 3 C, then cooled in desiccator and weighed to a nearest g. The samples were immersed in distilled water for 2 h at 23 C and immediately weighed to determine the absorbed water; process utilised for materials having a relatively high rate of absorption Mechanical testing Tensile tests were performed by means of an Instron Model 1122 testing system. Crosshead speed was 1 mm/min. Tensile strength at breakage

4 220 E.M. Salmoral et al. / Industrial Crops and Products 11 (2000) and percent elongation at breakage were measured using at least five specimens for each composition or treatment tested. The reported results are averages of the experimental values. Due to the limitations pointed by ASTM D-1708 we did not intend to measure the modulus. Tensile tests were performed after temperature and relative humidity of stabilisation of specimens. It was necessary to measure the moisture content of samples before mechanical testing below 1% because of the R.H. conditioned was different from the standard condition of 50% Statistic results The results were analysed by means of the Student s t-test. 3. Results In each case the starch employed was obtained from the same vegetal species and purified. The characterisation of the chemical structure showed that the white bean starch has a ratio of amylose to amylopectin structure of 70/30, while for the black bean the ratio is 95/5. The degree of branching points in the black bean starch was found to be 2.9. It was obtained from the relationship between the max and the percentage of 1,6- linked glucose. The 2.9 value founded in the black bean can be more accurately interpreted by comparison with the value of the degree of branching points of rabbit liver glycogen 9.4, phytoglycogen 7.1, amylopectin 5.7 and amylose of sweet corn maize 1.4 (Tolmasky and Krisman, 1987). This relations suggest the presence of an amylose with a relatively high degree of branching points. The absorption spectrum of the predominant glucopolysaccharide presents max=580 nm in the presence of CaCl 2 I 2 KI reagent in both cultivars. The preparation of blends for molding from defatted soy whole flour black and white bean whole flour was performed under the same conditions and proportions used for the protein preparation. When ultimate tensile strength of the plastic materials from whole flours of black and white beans are compared with those from the protein soluble fractions, it is found that the tensile strength (MPa) is significantly higher for protein materials (BP, P 0.001) and (WP, P 0.001) with respect to the whole flour materials (BWF) and (WWF). This effect was common to both varieties of beans ( Fig. 1A). When elongation at breakage is analysed, a notable difference between both varieties of beans can be observed. While the protein plastic compound of black bean (BP) reaches a high elongation respect of BWF, P 0.001, the white plastic bean variety shows better elongation in their whole flour compound WWF, P than the protein plastic compound (WP) (Fig. 1B). Moreover, the % elongation in the BP compound is seven times higher than WP compound, 0.01 P The water absorption of these Table 1 Water absorption of materials % a Blend Blend ba Blendi Black bean BWF BP White bean WWF WP a Black bean whole flour (BWF); black bean protein preparation (BP); White bean whole flour (WWF); White bean protein preparation (WP); Blend: protein preparation or defatted whole flour; 11.7% starch; 21.7% glycerol; 28% water; (Blend ba): blend with 3% boric acid; (Blendi): blend gamma irradiated to 50 kgy.

5 E.M. Salmoral et al. / Industrial Crops and Products 11 (2000) Fig. 1. Mechanical properties of plastic materials of soluble protein preparation of black bean (BP), soluble protein preparation of white bean (WP), black bean whole flour (BWF) and white bean whole flour (WWF). A: tensile strength expressed in MPa. B: elongation %. Blend: isolated or defatted whole flour +11.7% starch, +21.7% glycerol.

6 222 E.M. Salmoral et al. / Industrial Crops and Products 11 (2000) Fig. 2. Effect of boric acid. Mechanical properties of soluble protein preparation of black bean (BP), soluble protein preparation of white bean (WP), black bean whole flour (BWF) and white bean whole flour (WWF). Tensile strength expressed in MPa.: Elongation %. Blend. isolate or defatted whole flour +11.7% starch % glycerol +3% boric acid. same materials can be seen in Table 1. Whole flour materials BWF, 27% ( =1.0); WWF, 29% ( =2.0) absorb 20 5% more water than protein materials BP, 22% ( =0.45); WP, 21% ( = 0.69); in all cases water absorption was below 30% Boric acid treatment The effect on mechanical properties, tensile strength and elongation by the addition of boric acid in a concentration of 3% to the blends containing 11.7% starch and 21.7% glycerol are shown in Fig. 2. The result obtained show that neither the tensile strength nor the % elongation favourably influenced in plastic compounds based on black bean. There is also no positive influence in water absorption of these materials, as can be seen in Table 1. When the results of mechanical properties of white bean plastics materials are analysed, a different behaviour can be appreciated. The tensile strength only increases in the whole flour material from WWF, MPa (P 0.001). The % elongation resulted improved in the WWF from 1.8 to 5.6 (P 0.001); in WP from 2.5 to 3.5 (P 0.001), as can be seen by comparing Fig. 1B and Fig. 2. There was no positive influence in water absorption The gamma radiation The effect of gamma radiation on bean plastics can be seen in Fig. 3. By comparing with Fig. 1A, B tensile strength shows an increase in the case of black bean whole flours compound ( BWF) and white bean whole flours compound (WWF), (3 6 MPa, 0.01 P 0.001) and ( MPa, P 0.001), respectively.

7 E.M. Salmoral et al. / Industrial Crops and Products 11 (2000) Regarding percentage of elongation, only WWF was improved from 2.5 to 10.5, P The water absorption was reduced in both whole flour plastic compounds BWF, 17%, and WWF, 20%. 4. Discussion It has been demonstrated in Fig. 1 that the protein black bean compound (BP) reaches the higher percentage elongation and the higher tensile strength, which are not attained by the other materials studied. These results can be attributed to the chemical characteristics inherent in the cultivars under study. The interpretation requires having into account the remarkable difference in chemical composition existing between the soluble protein preparations BP, or WP and the defatted whole flours compounds BWF or WWF. These latter materials have 21.3 and 18% of cellulose content respectively, which is absent of the BP and WP materials. It should be remarked that the protein quality inherent to the vegetal species would have an influence on the gelatinization process considering that among proteins, the properties of hydration dampness or absorption and retention of water depend on the interactions protein water which depend also on ph and ionic strength. The much higher elongation in BP can be understand by the improved protein-protein interactions with better possibility of plasticization when the cellulose content is absent. However, the improved tensile strength, also suggests the influence of the typically quality protein of each cultivar, because of the WP compound has similar but reduced behaviour. Fig. 3. Effect of gamma radiation on the mechanical properties of protein soluble preparation of black bean (BP) and white bean (WP); black whole flour bean (BWF), and white whole flour bean (WWF). Tensile strength expressed in MPa. Elongation %. Irradiation of the blend at 50 kgy.

8 224 E.M. Salmoral et al. / Industrial Crops and Products 11 (2000) Moreover, the presence of lecithin in the soluble protein fraction based on black bean seeds would be contribute to the plasticization process (actually in study at the laboratory). It is known besides, that the starch structure determines the properties of starch material, such as stability, degradability, texture, mechanical and thermal properties. There is a diversity of molecular and supramolecular structures in processed starch materials depending on starch source, particularly amylose/amylopectin ratio, moisture and plasticizer content (Van Soest, 1996). The starches in study possess different ratio of amylose/amylopectin, 70/30 and 95/5, in white and black bean respectively. This work shows a highly hydrophilic carbohydrate chains in the case of predominant amylose structure that would have better influence on the gelatinization process in the presence of water and consequently in the mechanical properties of the BP plastic material. Irradiation of blends produced an appreciable effect regarding tensile strength and water absorption capacity of whole flour products. We did not found an effect of plasticization that would have resulted in case of degradation of starch molecules. This effect would have been noticeable as an improvement in elongation. The cellulose material present in the defatted whole flour could be affected by radiation through breaking of chains and introducing radiolytic products. Both effects would enhance the reactivity of the cellulose material (Stepanik et al., 1998). The improvement in tensile strength and water absorption observed could be attributed to interactions among cellulose chains, proteins and the amylopectin starch structure. Fig. 2 shows that boric acid only has chemical effect on materials based on white beans; this effect improves the mechanical properties, which was associated with the presence of different kind of starch. The plastic products of beans studied absorb not more than 30% of water. These values are four times lower than that of the soy whole flour and two times lower than the protein soy isolate respectively, treated in similar conditions. (Salmoral et al., 1999). At the moment of this presentation biodegradability essays have begun at our laboratory. Further experiments with the purpose to improve the presented plastics are to be carried on, among them the elucidation of the protein quality of the beans. 5. Conclusion Black and white bean leguminous under study proved to be potential raw materials for manufacturing biodegradable plastics by compression moulding. The plastic materials obtained from soluble protein preparations without cellulose content and those formed by whole flours have different mechanical properties in some cases. This result may be attributed to the association protein glucopolysaccharide (amylose or amylopectin) in presence of glycerol and water as plasticizers. So, the plastic material based on black bean, made with a glucopolysaccharide characterized by its prevailing amylose structure, is outstanding because its elongation as well as its tensile strength are higher than all the studied materials and because of its low ability to absorb water. It could be said that, in the conditions employed in this study, black bean produced plastic materials with better properties than all soybean materials, such as protein isolate and whole soy flour. On the other hand, plastics based on white bean with glucopolysaccharide characterised by having higher amylopectin content had poorer mechanical properties and higher water absorption. Nevertheless, the mechanical properties could be improved, the same as water absorption, by including boric acid in the blends of black bean legume. However there was a slight improvement in the mechanical properties of whole flour white bean. The effect of gamma radiation was important only in the plastic materials with cellulose content (BWF, WWF), improving their tensile strength and reducing to 17 and 21% the water absorption, respectively.

9 E.M. Salmoral et al. / Industrial Crops and Products 11 (2000) The protein soluble fractions resulted better than the defatted whole flours in both bean varieties. The economic advantage is that we can utilise in the plastic moulding process, the destroyed seeds which are habitually rejected as wastes. Acknowledgements Research and Doctorate Department, Engineering Faculty of Buenos Aires University,for financial support. For provision of seeds: to Eng. L.F. Medina and Experimental Stations of National Institute of Agricultural Technology ( INTA). For lyophillization of samples to National Institute of Seeds and Matias Langan. References ASTM, ASTM Standard D-1708 Standard test method for microtensile tests of plastics, American Society for Testing and Materials, Philadelphia. ASTM, ASTM Standard D-570 Standard test method for water absorption of plastics, American Society for Testing and Materials, Philadelphia. Kinsella, J.E., Functional properties of soy proteins. Am. Oil Chem. Soc. 56, Lowry, O.H., Rosebrough, N.J., Ferr, A.L., Randall, R.J., Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, Pruzinek, J., Hola, O., Starch degradation by irradiation. J. Radioanal. Nucl. Chem. Lett. 118 (6), Ressouany, M., Vachon, C., Lacroix, M., Irradiation dose and calcium effect on the mechanical properties of cross -linked caseinate films. J. Agric. Food Chem. 46, Salmoral, E.M., Cura, J.A., Pagano, E., Tolmasky, D., Lepek, V., Favret, E., et al., Comparative studies of the -1,4-1,6 glucopolysaccharides from crops of commercial interest. An. Asoc. Quim. Argent. 81 (2 3), Salmoral, E.M., Gonzalez, M.E., Mariscal, M.P., Medina, L.F., Plastic compound of soy. Fifth Latin American Symposium of Polymers, Chile. Salmoral, E.M., Gonzalez, M.E., Mariscal, M.P., Medina L.F Biodegradable plastics made from chickpea and soy. Fourth European Symposium on Industrial Crops and Products together with the Sixth Symposium on Renewable Resources for the Chemical Industry, Bonn, Germany. Salt, D.J., Leslie, R.B., Lillford, P.J., Dunhill, P., Factors influencing protein structure during acid precipitation. A study of soy proteins. Eur. J. Appl. Microbiol. Biotechnol. 14, Stepanik, T.M., Rajagopal, S., Ewing, D., Whitehouse, R., Electron processing technology: a promising application for the viscose industry. Rad. Phys. Chem. 52, Tolmasky, D., Krisman, C.R., The degree of branching in -1,4-1,6 linked glucopolysaccharides is dependent on intrinsic properties of the branching enzymes. Eur. J. Biochem. 168, Van Soest, J., Starch plastics:structure, property relationship. Thesis, University Utrecht. Vizgarra, O., Tuc 390 and Tuc 500: two new varieties of black bean for the Northwest of Argentina. J. Ind. Ag. Tucumán 72 (1 2), Yu, J., Che, S., Gao, J., Zheng, J., Lin, T., A study on the properties of starch/glycerine blend. Starch/Starke 50 (6),