Post-harvest stability of latex in different sizes of
guayule branches
Katrina Cornish
a,*, Mary H. Chapman
a, Jenny L. Brichta
a,
Stephen H. Vinyard
b, Francis S. Nakayama
baUSDA-ARS,Western Regional Research Center,800 Buchanan Street,Albany,CA 94710, USA bU.S. Water Conser6ation Laboratory,Phoenix,AZ 85040, USA
Received 26 July 1999; accepted 30 October 1999
Abstract
Commercial development of hypoallergenic latex fromParthenium argentatum(Gray) for the manufacture of latex medical and household goods, is hampered by the lack of information on latex stability in the harvested shrub prior to processing. In this paper, we investigate the effect of post-harvest storage on extractable latex content of guayule branches. We found that harvested guayule branches can be stored at 4°C for at least 2 weeks without compromising latex yield, provided that dehydration does not occur. When stored hydrated at 4°C, latex levels declined between 2 and 5 weeks in all branch sizes with the most latex lost from the smallest branches and the least from the medium ones. The latex levels declined due to a combination of conversion of latex into solid rubber in situ and rubber degradation, depending upon the branch size. Overall rubber degradation from the latex and solid rubber pools in the branches was substantial in the smallest branches, but was not seen in the largest. Latex levels declined more quickly at warmer temperatures, even when the branches were hydrated, and were adversely affected by even slight dehydration. Published by Elsevier Science B.V.
Keywords:Guayule; Hypoallergenic latex;Parthenium argentatum; Storage
www.elsevier.com/locate/indcrop
1. Introduction
Parthenium argentatum (Gray), commonly known as guayule, is a perennial, woody shrub native to the Chihuahuan desert of the United States and Mexico (Whitworth and Whitehead, 1991). Current commercialization efforts are
based on the extraction of intact rubber particles from guayule shrub in an aqueous suspension, and their subsequent purification as a high qual-ity, low protein, hypoallergenic latex (Cornish, 1996, 1998). This latex provides a safe alternative source of natural rubber latex for patients suffer-ing from life-threatensuffer-ing Type I latex allergy (Carey et al., 1995; Siler and Cornish, 1994; Siler et al., 1996). Guayule latex has been characterized (Schloman et al., 1996), successful prototype latex medical and consumer products have been made * Corresponding author. Tel: +1-510-559-5950; fax: +
1-510-559-5663.
E-mail address:[email protected] (K. Cornish)
(Cornish et al., 1996; Cornish and Lytle, 1999), but full commercialization has not yet been real-ized. To expedite this goal, factors affecting the stability of latex rubber in harvested guayule shrub prior to processing, must be understood in order to establish and optimize post-harvest stor-age conditions for the shrub, so as to maximize latex yield.
In this paper, we investigate the effect of dehy-dration, temperature, length of storage, and branch size on the yield of latex, rubber and resin components in guayule shrub.
2. Materials and methods
2.1. Plant material
Branches were harvested from mature guayule shrub, grown at different field locations in Ari-zona. Branches were dipped into 1% aqueous ascorbate (an anti-oxidant), sealed in plastic to prevent dehydration, iced, and shipped overnight to Albany, CA.
All harvested branches then were sorted into three branch diameters, which approximated the relative age of the different tissues: small B0.5, medium 0.5 – 1.0, and large \1.0 cm in diameter. The smallest branches contain very little woody material and represent the current year’s growth. The medium branches are over a year old, contain a woody core, and have experienced one winter season. The largest branches are at least 2-years old, contain a substantial woody core, and have experienced at least two winters. The branches were stored in sealed plastic bags, until processed.
2.2. Latex extraction
Filtered homogenates were prepared according to the Waring blender method previously de-scribed (Cornish et al., 1999) in which the samples are ground for 1 min in 1:2 w:v shrub:extraction buffer (0.2% ammonia, as NH4OH, 0.1% Na2SO3, pH 10), filtered through a 1 mm steel mesh, and the plant material (bagasse) retained by the filter reground for 1 min in the same volume of extrac-tion buffer used before. The homogenate from
this second grind also was filtered and the two filtrates were pooled for latex quantification. Thus, this procedure entails 2×1-min grinds and a total of 1:4 w:v of shrub to extraction buffer. In some experiments, the bagasse was reground and filtered two more times for a total of 4×1-min grinds. In these experiments, the filtrates were not pooled and samples of each filtered homogenate and retained bagasse were analyzed for latex (in the homogenate) or residual rubber and resin (in the bagasse). Homogenates usually were made separately from the different branch sizes, except in the dehydration and temperature experiments where branch sizes were pooled before homoge-nization. Homogenates were stored in sealed glass bottles at 4°C until quantification of their latex content, which occurred within 3 days.
2.3. Latex quantification
Latex content of all homogenates was quantified using the 1-ml quantification method previously described (Cornish et al., 1999), where 1-ml aliquots of homogenate are centrifuged to float the latex, which is then coagulated with glacial acetic acid, harvested, rinsed, dried and weighed.
2.4. Dehydration
One day after harvest, fully hydrated branch samples (100 g, \0.5 cm in diameter) were dried to different moisture levels, ranging from 100 to 51% relative water content (RWC) using 24°C (room temperature) or 40°C air-drying. The fresh weight of the shrub after shipping to California (1 day after harvest, see Section 2.1.) was considered to reflect 100% RWC. Completely dried shrub is 0% RWC. The largest branches took 24 h to achieve 51% RWC. As each sample reached the desired % RWC it was sealed in plastic to prevent further water loss, stored at 24°C, and processed 7 days after the original harvest.
2.5. Temperature dependence
buffer to maintain hydration, and stored at 4, 24 or 40°C for up to 2 weeks until pro-cessed into homogenate for latex quantifica-tion.
2.6. Rubber and resin analysis of shrub and bagasse
The rubber and resin contents of samples of original shrub and of the bagasse resulting from the latex extraction method (Section 2.2.) were determined by extraction with acetone (for the resins) and cyclohexane (for the rubber) (Black et al., 1983). When the latex and residual rubber yields were to be directly compared (as in Fig. 5), the bagasse rubber concentrations were corrected to reflect the original branch weight ground because the non-rubber solids smaller than 1 mm generated by grinding are lost to the ho-mogenate during the filtration step and are not retained in the bagasse fraction (Cornish et al., 1999).
3. Results and discussion
3.1. Effect of dehydration on extractable latex in har6ested guayule branches
Dry shrub has no extractable latex because the rubber particles are no longer suspended in the aqueous cytoplasm. This dried rubber can be extracted with appropriate organic solvents (see reviews in Whitworth and Whitehead, 1991), but the resultant solid rubber cannot be used to man-ufacture high value hypoallergenic latex products, which require the rubber to be in latex form (a colloidal suspension of a polymeric material in a liquid system mostly aqueous in nature and, in the case of guayule latex, an aqueous suspension of rubber particles). A method to reconstitute a col-loidal suspension from the solvent-extracted solid rubber has been described (Schloman et al., 1997) and, as expected, this reconstituted (or ‘semi-syn-thetic’) rubber latex contained very little protein and was hypoallergenic with respect to Type I latex allergy. However, in contrast to the native guayule latex obtained from the aqueous extrac-tion method, there has been no demonstraextrac-tion that high quality dipped film products could be successfully made from this material (with re-quired stress and strain characteristics and viral barrier properties, etc.). Furthermore, the solvent-extracted rubber previously had been shown to not be commercially competitive with tropical rubber for the solid rubber markets. Reprocessing into a reconstituted latex would undoubtedly re-quire an unknown but additional cost. Thus, even if high quality products could be made from this material they would be considerably more expen-sive than the native latex products.
Latex extraction from guayule shrub is entirely dependent upon the presence of discrete rubber particles suspended in the aqueous cytoplasm of the parenchyma cells whereas, as discussed above, dry shrub has no extractable latex. Therefore, we investigated the effect of dehydration of guayule branches on extractable latex yield. Latex yield began to decline under slight dehydration (RWC=82.2%, equivalent to a 10% loss of fresh weight) and very little latex remained above a RWC of 51% (equivalent to a 25% loss of fresh Fig. 1. Effect of dehydration on extractable latex content of
Fig. 2. Effect of temperature on extractable latex content of hydrated guayule branches (a mixed sample of all three sizes) stored for up to 2 weeks before processing with 2×1-min grinds. The three temperature regimes were imposed after harvest and storage for 1 day at 4°C. Each value reflects the mean latex content of three branch samples 9S.E. (Each value in the mean was the mean of six determinations of the latex content of each branch homogenate.)
weight (Fig. 1). The dry weight of completely dried branches (RWC=0%) was 44% of the hy-drated weight (or after a 56% loss of fresh weight). It is clear that all of the extractable latex was lost well before all of the moisture in the tissue. The significant effects on latex content of mild dehydration may reflect heterogeneity of the branch tissue — perhaps a subset of cells near the cut ends of the branches, or near the branch surface, severely dehydrated coagulating the rub-ber contained within, whereas the remaining cells were much less affected. An earlier investigation also showed adverse effects of dehydration on total rubber and extractable latex levels (Nakayama and Coates, 1996), but due to the different methods employed, the results cannot be directly compared. Nevertheless, it is clear that the harvested guayule shrub must be protected from water loss while waiting to be processed to prevent substantial latex losses.
3.2. Effect of temperature on extractable latex in har6ested guayule branches
Preliminary storage experiments on hydrated guayule branches (43.9% dry weight), sealed in plastic bags to prevent dehydration, demonstrated that extractable latex content also was sensitive to post-harvest storage temperature (Fig. 2). Refrig-eration at 4°C, however, did prevent latex loss for up to 2 weeks when dehydration was avoided. Extractable latex levels rapidly declined at the higher storage temperatures, with about half the latex lost after 5 days at 24°C and more than 90% after 5 days at 40°C.
3.3. Stability of latex in har6ested guayule
branches
Latex stability then was examined in three dif-ferent sizes of guayule branches: B0.5 cm diame-ter, 40.5% dry weight; 0.5 – 1.0 cm diamediame-ter, 43.9% dry weight; \1.0 cm diameter, 48.5% dry weight. The branches were stored at 4°C in sealed plastic bags to prevent dehydration of the tissue. No significant decline in extractable latex was observed in any of the branches during 2 weeks of storage (Fig. 3).
Fig. 4. Extractable latex expressed as a percentage of the total rubber contained in branches of three diameters:, B0.5 cm;, 0.5 – 1.0 cm;, \1.0 cm. Branches were stored in sealed plastic bags at 4°C for 2 or 5 weeks before processing with a series of 1-min grinds. Each value is the mean of three determinations. For clarity, the standard errors (all 55%) are not shown.
In a different batch of branches, also stored at 4°C, the proportion of the total rubber ex-tractable as latex after 2 weeks was quite similar in all three branch sizes, ranging between 68 and 79% (Fig. 4a). However, after 5 weeks, only the medium sized branches were still in this range, and had dropped slightly (Fig. 4b). The largest branches showed the greatest decline, between 2 and 5 weeks, with the proportion of rubber ex-tractable as latex dropping to about 50%. The different rates of decline were not the result of dehydration (see Fig. 1) because all the branches, which were wetted before sealing into plastic bags, absorbed water during storage (Table 1). Further-more, the relative amount of extractable latex that could be obtained by the first and second 1-min grinds was lower after 5 weeks of storage than after 2 weeks (Fig. 4). This suggests that 3× 1-min grinds might best be used on branches stored for more than 2 weeks for effective latex extrac-tion. It has previously been shown that 2×1-min grinds release at least 90% of the extractable latex from guayule branches while 4×1-min grinds release at least 99% (Cornish et al., 1999). Nonetheless, the expense of a third or fourth grind, from a commercial perspective, may not be worth the additional latex yield obtained.
Similarly, in this investigation, 2×1-min grinds extracted most of the latex rubber from the
guayule branches (Fig. 5A and D). However, we observed that the extractable latex content de-clined substantially in all three branch sizes be-tween 2 and 5 weeks, with the smallest branches being most adversely affected (Fig. 5A and D). The decline in latex in these small branches was partially off-set by an increase in the solid rubber component still retained by the bagasse (deter-mined by solvent extraction) (Fig. 5B and E), presumably reflecting in situ coagulation of the rubber particles, and possibly gel (insoluble cross-linked polymer) formation. A loss in total rubber was also apparent (Fig. 5C and F), by it was not possible to determine whether the loss reflected degradation of either or both the latex and solid rubber pools in the branches.
In contrast, the largest branches had very little change in total rubber content at 5 weeks
com-Table 1
Water content (%) of guayule branches of three different sizes, stored for 2 or 5 weeks calculated as [(fresh weight−dry weight)/fresh weight]×100
Storage time (weeks) Branch diameter (cm)
\1.0
B0.5 cm 0.5–1.0
48.9
2 53.2 55.5
60.0 60.6
Fig. 5. Extractable latex, residual rubber (in the bagasse), and total rubber in guayule branches of three diameters:, B0.5 cm;
, 0.5 – 1.0 cm;,\1.0 cm. Branches were stored in sealed plastic bags at 4°C for 2 or 5 weeks before processing with a series of 1-min grinds. All values are based upon the amount of branch material originally processed, and total rubber values are the sum of the latex and bagasse rubber determinations. Each latex value is the mean of three determinations, each bagasse rubber value is a single determination.
pared with 2 weeks (Fig. 5C and F); the decline in extractable latex content (Fig. 5A and D) was balanced by a concomitant increase in the solid rubber bagasse component (Fig. 5B and E). Also, it appeared that the extraction of total rubber from the largest branches, that were not homoge-nized for latex extraction, was incomplete (Fig. 5C and F, () compare grind number 0 with grind numbers 1 – 4) and similarly for resin extrac-tion, at least after 2 weeks of storage (see Fig. 6B ()).
The smaller loss of latex from the medium branches, between 2 and 5 weeks of storage (Fig. 5A and D), was paralleled by a loss of total rubber (Fig. 5C and F) with very little change in
the solid rubber content retained by the bagasse (Fig. 5B and E). Thus, either the latex itself was partially degraded, without first coagulating in situ to form solid rubber or, alternatively, a simi-lar quantity of already solid rubber, to the amount of latex coagulated between 2 and 5 weeks, was degraded. Nevertheless, irrespective of storage time, the medium-sized branches ()
con-sistently had the highest content of extractable latex, at almost 100 mg/g (10%) on a dry weight basis after 2 weeks of storage (Fig. 5A) and 67 mg/ml (6.7%) after 5 weeks (Fig. 5D).
and E). However, less resin was found in the medium size branches after 5 weeks than after 2 weeks, which may indicate degradation during storage. It was also apparent that more of the rubber component became much less extractable as latex after 5 weeks, which was especially notice-able in the largest branches (Fig. 6A and D). All three branch sizes contained a similar ratio of rubber and resin in their tissues prior to grinding (Fig. 6C and F, grind number is 0). However, the rubber to resin ratio dropped substantially during grinding (Fig. 6C and F, grinds 0 – 4). Thus, the amount of resin extracted from all three sizes of guayule branch was disproportionally less than the amount of latex extracted. Also, it was appar-ent that less resin was left in the bagasse the
greater the grind number (Fig. 6B and E). The fate of this resin loss should be investigated to determine if it reappears as a latex contaminant.
4. Conclusions
Latex yield in harvested guayule branches is sensitive to the degree of dehydration and losses are noticeable at 80% RWC. Very little ex-tractable latex remains at 50% RWC, probably due to coagulation of the latex in situ. Hydrated guayule branches can be stored for at least 2 weeks at 4°C without compromising latex yield, but higher temperatures appear to cause latex degradation. When hydrated branches were stored
at 4°C for 2 – 5 weeks, their latex content declined due to a combination of conversion into solid rubber and degradation, with latex losses greatest in the smallest and largest branches.
Acknowledgements
Research was supported, in part, by USDA-CSREES Fund for Rural America, Grant No. 97-36200-5181. The authors thank Drs D.J. Scott, A.E. Stafford and Ms C.A. Hudson for their critical reviews of this research.
References
Black, L.T., Hamerstrand, G.E., Nakayama, F.S., Rasnik, B.A., 1983. Gravimetric analysis for determining the resin and rubber content of guayule. Rubber Chem. Technol. 56, 367 – 371.
Carey, A.B., Cornish, K., Schrank, P.J., Ward, B., Simon, R.A., 1995. Cross reactivity of alternate plant sources of latex in subjects with systemic IgE mediated sensitivity to He6ea brasiliensis latex. Ann. Allergy Asthma Immunol. 74, 317 – 320.
Cornish, K., Lytle, C.D., 1999. Viral impermeability of hy-poallergenic, low protein, guayule latex films. J. Biomed. Mater. Res. 47, 434 – 437.
Cornish, K., Bader, H.F., Lytle, C.D., 1996. Manufacture and testing of guayule latex products. In: Proceedings of the International Meeting of the AAIC, San Antonio, TX, p. 36 Abstract.
Cornish, K., Chapman, M.H., Nakayama, F.S., Vinyard, S.H., Whitehand, L.C., 1999. Latex quantification in guayule shrub and homogenate. Ind. Crops Prod. 10, 121 – 136.
Cornish, K., 1996. Hypoallergenic natural rubber products fromParthenium argentatum(Gray) and other non-He6ea brasiliensisspecies. U.S. Patent No. 5 580 942.
Cornish, K., 1998. Hypoallergenic natural rubber products fromParthenium argentatum(Gray) and other non-He6ea brasiliensisspecies. U.S. Patent No. 5 717 050.
Nakayama, F.S., Coates, W., 1996. Storage effects on rubber content of laboratory- and field-prepared guayule shrub. In: Princen, L.H., Rossi, C. Jr (Eds.), Proceedings of the Third International Conference on New Industrial Crops and Products, 25 – 30 September 1994, Catarmarca, Ar-gentina. Versa, East Peoria, IL, pp. 243 – 246.
Schloman, W.W., Wyzgoski, F., McIntyre, D., Cornish, K., Siler, D.J., 1996. Characterization and performance testing of guayule latex. Rubber Chem. Technol. 69, 215 – 222. Schloman, W.W. Jr, McIntyre, D., Siler, D.J., Stumpf, D.K.,
Hoffmann, J.J., 1997. Semisynthetic guayule latex with reduced allergenicity. Ind. Crops Prod. 7, 27 – 36. Siler, D.J., Cornish, K., 1994. Hypoallergenicity of guayule
rubber particle proteins compared toHe6ealatex proteins. Ind. Crop Prod. 2, 307 – 313.
Siler, D.J., Cornish, K., Hamilton, R.G., 1996. Absence of cross-reactivity of IgE antibodies fromHe6ea brasiliensis latex allergic subjects with a new source of natural rubber latex from guayule (Parthenium argentatum). J. Allergy Clin. Immunol. 98, 895 – 902.
Whitworth, J.W., Whitehead, E.E. (Eds.), 1991. Guayule Nat-ural Rubber: A Technical Publication with Emphasis on Recent Findings. Guayule Administrative Management Committee and USDA Cooperative State Research Ser-vice, Office of Arid Lands Studies, The University of Arizona, Tucson, AZ.
