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Using vegetable tannin to preserve natural rubber latex and to decrease or suppress its allergic action.

Vegetable tannins are within a large category of natural polyphenols found in many plants and derived products (ref. 1). Their versatile molecules allow for their use in a multitude of industrial applications, like skin tanning, well drilling lubrication (ref. 2), flocculants (ref. 3) and particle settlers in water treatment (ref. 4), and wood adhesives (ref. 5). Leather production, by far the most prominent (ref. 6) tannin industrial utilization, has made use of these natural extracts from various plant sources for many centuries.

Flavonoid molecules from the condensed type of tannin, shown in figure 1, are very characteristic for their large number of hydroxyls, which through hydrogen bonds (ref. 7) are the chemical basis for their industrial uses, especially the tanning of the skin collagen, as illustrated in figure 2.

As is widely known, proteins are at the center of two major problems in natural rubber latex (NRL) technology, namely allergies caused by latex artifacts, such as in gloves and condoms, and the vulnerability of latex to biological deterioration. Although the importance of the difficulties emerging from both problems has demanded research efforts for decades, complete solutions are still to be found (ref. 9). Notwithstanding the well-known effect of vegetable tannin on protein molecules, the basis of the secular tanning process in leather production, the approach of treating latex rubber in natura with tannin solution has not been recorded in the specialized literature, if it has ever been tried.

As proposed by the interaction mechanism in figure 2, in the transformation of hide to leather, hydroxyl groups become linked to collagen protein molecules, which, after this treatment, are not susceptible any longer to microbiological attack. Furthermore, these changed molecules lose their ability to react as proteins, since they are bonded to tannin by strong hydrogen bonds in a very stable and irreversible way, as confirmed by the leather stability (ref. 10). If they are blocked against bacterial attacks, it is expected that these resulting protein-tannin complexes are not able to react in highly specific interactions (ref. 11), such as allergic sensitization.

The whole of this work has taken four years, including the execution of various experimental plans and several variables which were measured and followed; and the full work resulted in a doctoral thesis, and a patent referring to the subject was granted.

Methodology

Materials

The main research item, latex in natura (LIN), was collected at a plantation of Hevea brasiliensis, clone RRIM 600, used as normal field latex with about 30% dry matter. It should be recorded that the latex was abnormally unstable for the collection period, at the beginning of the production season, combined with the unusual high temperature observed on the collection day. This condition would strongly influence the stability of samples, as will be seen. Tannin was used as an aqueous solution at 25% (w/w), prepared the day before from the powder sold by Tanac S.A. as Weibull vegetable tannin, extracted from black acacia bark (Acacia meamsii De Wild). Two surfactants were used as received, including an ionic type, sodium lauryl ether sulfate (SLES or laureth), commercial aqueous solution with 23% active content (w/w), from Garden Ltd.; and a nonionic surfactant, Renex, commercial aqueous solution with 27% (w/w) active content of nonylphenol ethoxylate, from Oxiteno. Borax, as a mild bactericide, was used at 5% (w/w) aqueous solution. Potassium hydroxide was used at 2 mol. [L.sup.-1] aqueous solution. Ammonia, N[H.sub.4]OH, was used as an aqueous solution of 28% (w/w).

Sample formulations of natural rubber latex (NRL) The latex formulations were prepared as shown in table 1.

Analysis and measurements

Physical and biological stability of latex with time

Before measurements and analysis, samples were observed to evaluate latex general aspects. The following determinations were made on the samples, right after preparation and after different elapsed times:

* Smell score was assigned to samples by at least two experienced observers. The score scale was set from zero to ten: Zero means the sample smelled rotten, and ten means the sample had the best fresh and pleasant latex scent. At the intermediate point five, the sample starts changing from fragrant to uncomfortable.

* pH measurement was done in the usual way, in one reading, using an electrode model designed for more viscous media.

* Zeta potential was determined with samples diluted 10,000 times in water, using the Zetasizer Nano Z90 from Malvern Instruments, with excitation at 632.8 nm.

Protein allergenic degree

Two aliquots (12 mL) of samples that were still in liquid form were centrifuged in Hermle Z 32 HKC equipment at 8,000 rpm, at 16[degrees]C for 60 minutes. The serum and the bottom fraction were withdrawn with a long needle syringe, and the cream was re-diluted with water plus ammonia for the controls, and water plus tannin for the tannin treated formulations, in such a way as to reproduce approximately the initial conditions. Latex cast thin films obtained from these samples were prepared in an oven under vacuum at 60[degrees]C for 24 hours, at three stages, including non-centrifuged and after one and two centrifugations. Samples of around 3 grams obtained from these films were sent to the Tun Abdul Razak Research Center, in the United Kingdom, for determinations of the concentration of specific allergenic proteins Hev b5 and Hev bl3, following the ELISA (enzyme-linked immunosorbent assay) for latex.

Results and discussion

A theoretical model proposal to explain tannin action on NRL Before presenting experimental results, a theoretical model of a colloid particle protection shield is proposed to explain the tannin activity on NRL. A possible theoretical construction can be suggested in which tannin complexes with proteins surrounding the particles, creating a kind of protective shield, as proposed schematically in figure 3. This kind of protection avoids the colloid coagulation by acting through steric hindrance rather than ionic repulsion, normally related to the colloid stability. As the tannin molecule has plenty of hydroxyls, part of them are used for the H-bonds with the protein, but others, on the opposite side, are available for holding water molecules, making the shield even stronger.

Measurements following latex preservation with time

Samples were observed eight times over a period of 17 days. From preliminary experiments, this period was considered sufficient to verify if the tannin samples succeeded in preserving the NRL against decay. One last reading was made only for the analysis of volatile fatty acids (VFA) for the treatments HA and TBR.

From the observation of the general characteristics, it could be observed that control samples were white in color, did not stain, and formed neither layers nor deposits. However, small spots appeared on the surface and those samples collapsed by coagulation in short time, depending on the percentage of the ammonia: Two days with low ammonia, to around 16 days for the high ammonia. Samples with tannin showed a light pink color and did not coagulate for the entire period of observation, 17 days. Some tiny spots on the surface or stains on the walls appeared as the tannin treated latex aged. The tannin latex formed red deposits on the bottom of the sample container, signaling the characteristic tannin red color. As the remaining volume of samples was kept under observation, it was possible to observe that the tannin formulations were still fluid, with no sign of coagulation after a period of four months, whereas the controls with no tannin were all coagulated. This observation confirmed previous non-published experiments. It should be explained here that the rather unusual short period of NRL with ammonia, which normally stands for several months, was due to the initial period of the rubber production season, when NRL is very unstable, coinciding with the high temperature, around 38-40[degrees]C, on the day of latex collection and experimental set-up.

Smell tests

Table 2 shows the smell recordings for the samples. Although the experiment was influenced by unusual critical conditions of latex instability, as just explained, results clearly indicate differences between the treatments. Sample LA had a very short life and coagulated on the third day of reading. Samples LAB and HA had a strong ammonia vapor, which precluded the smelling test. However, despite their higher ammonia concentration, both coagulated before the reading at 408 hours (17 days). Normally, NRL formulations with low or high ammonia do not coagulate spontaneously for long periods. The unusual short life of these ammonia treatments was due to the bad conditions for the latex itself, as mentioned before. However, as this condition affected all treatments and the experiment was conducted under the same severe situation for all treatments, the results highlight the good performance of the tannin samples.

The smell test is naturally subjective, but it provides a clear and straightforward result, which, when taken in a set of results of various readings, can be a simple and strong tool of analysis, as happened in the present case. From the table, it is easy to observe the treatments which failed in preserving the latex, differing from others that presented good results, like the ones based on tannin. Comparing both tannin treatments, it is possible to differentiate the two surfactants: The ionic, in sample TBL, starts in good condition and continuously decreases its score; whereas the non-ionic agent in sample TBR continues with a good smell until the end of this testing sequence. However, both samples remained fluid on the laboratory bench even after several months.

The smell of latex deterioration reflects the release of volatile fatty acids (VFA) as a result of bacterial attack. If tannin is blocking the proteins against bacterial decay, why is there production of VFA? The reason is simple: Tannin is not complexing other organic materials, such as lipids, carbohydrates and other components of the rich organic medium, which is the latex, all of them susceptible to bacterial degradation. That is the reason for the presence of a mild bactericide in the formulation.

Measurements of pH

The set of pH measurements over time is represented in figure 4. As already mentioned, the latex in natura was very unstable, as can be seen from the LA sample, which was very unstable even with ammonia, reflected in its high initial pH (9.44) and coagulation on the third reading (54 hours). Treatments LAB and HA, the other two non-tannin control samples, coagulated before the 17th day of reading. Sample LAB remained fluid for a longer time than LA, and the same period as HA, despite exhibiting a lower pH of 8.78 at the beginning of the experiment. This behavior can be ascribed to the presence of the bactericide.

It is worthwhile to note that all control samples presented a consistent pH decrease going themselves through collapse and coagulation, while both tannin treatments remained fluid at low pH values in which normally the latex should coagulate. For both tannin samples, there was a sharp pH decrease in the beginning stage, followed by a steady period, which may mean that tannin is protecting the proteins; but there are other digestible compounds like sugars and phospholipids that are not being complexed by polyphenols, which supports the view that tannin is making the difference, providing more stability to the colloid. This kind of observation from the objective measurement of pH comes to strengthen the model of the colloid particle protection shield, which is acting through the mechanism of steric hindrance rather than ionic colloid protection, although it is obviously necessary to do additional work and provide more details in its support.

Evolution of zeta potential with time

As can be observed from figure 5, all zeta potential measurements are negative, ranging from -32 to -29 mV, which can be expected for colloids, where rubber particles are surrounded by negatively charged proteins and phospholipids. As time elapses, zeta potentials pass through a minimum during the first 100 hours, then start increasing to reach a steady value after 200 hours. The first observation can be directed towards sample LA: It starts with high colloid protection, -36.4 mV, after 30 hours decreases this value to -29.0 mV, and then coagulates. Samples LAB and HA coagulated with zeta potential around -25 mV and -29 mV, respectively. So the region of -25 mV to -30 mV is critical and can be taken as a possible limit range before coagulation takes place in the systems under study, when and if the protection is mainly based on single ionic protection. Sample TBR, in the second part of the graph, exhibits zeta potential between -26 mV and -24 mV. This whole interval is below the limit of -29 mV and within the limit range observed above, which means that TBR should be coagulated itself, since electrostatic particle stabilization alone would be unable to keep the colloid stable. However, the TBR sample stayed in the liquid state during the 17 days of observation and displayed the best smell score. This unusual behavior for this latex also comes in support of another kind of colloid protection, reinforcing the shield model proposed.

Protein allergenicity quantification

Table 3 shows results determined by the Tun Abdul Razak Research Center (TARRC) in Hertford, U.K., based on the ELISA for two of the most studied latex allergenic proteins, Hev b 5 and Hev b 13. By doing these determinations, the attention was driven to search for differences between the ammonia sample, HA, and the tannin one, TBR, which had pro vided the best set of results in the first part of the experiments. On the Hev b 5 results, three of five results for the tannin sample fall below the limit of detection for the method (TBL/ NC, TBR/NC and TBL/C1), which is not observed for the ammonia sample, and which adds more signs that tannin is possibly acting to decrease allergenic action.

However, the measurements for the Hev b 13 do not provide a vision so straightforward. With no centrifuging, the result for TBR is double the result for the ammonia sample; but this pattern reverses in the two centrifuging stages to become much lower in the last one, with two centrifugations, where the ammonia sample presented a concentration of 50 [micro]g of Hev b 13 per gram of dried latex film, almost three times that of the tannin sample result of 17 [micro]g/g. These very clear results and their exchange of positions can be understood if one considers the place where Hev b 13 is present in the latex colloid. As published in the specialized literature (refs. 12 and 13), part of this protein is located inside the lutoid particles, where it is likely that tannin cannot reach those molecules to complex. However, in the centrifuging process, a great part of the lutoids, as heavier particles, is eliminated, but, possibly, a small part of them is destroyed by the strong shearing forces in the centrifuge, releasing the allergenic proteins to the serum (ref. 14), where they can then be complexed by tannin, resulting in the observed results for the ELISA. This fact can also be understood to support the proposal that the polyphenolic tannin is complexing proteins, in general, including the allergenic ones. Although there is plenty of space for other necessary experiments to confirm this tannin action to decrease or to eliminate latex allergenicity, the results provided by TARRC analysis open a new research approach to deal with this important subject.

Discussion about the model of tannin-protein particle shield

It is worthwhile to make it clear that not all results produced during the long period of experiments are presented here, as they were published (in Portuguese) as the author's doctoral thesis. However, for the sake of the rationale, all of the main conclusions are referred to in the text below.

The objective data produced as experimental results, which are to be taken in support of the view that tannin is providing structurally positive changes in the NRL in the two proposals, namely latex bio protection and a decrease in allergenicity, are as follows:

1) A field hevea latex, uncommonly unstable, was stabilized based on treatments with tannin, a mild bactericide, an ionic or non-ionic surfactant, and potassium hydroxide.

2) While controls with ammonia lasted less than 17 days, tannin samples were stable and fluid even after more than five months of observation on a laboratory bench scale.

3) Volatile fatty acid release, resulting from latex degradation, is much lower for tannin treatments than for the controls.

4) As a consequence, the smell of tannin-treated samples is reasonable after months of aging, while controls with ammonia presented a strong odor, characteristic of bacterial degradation of organic matter.

5) Tannin samples' pH values were in an interval where NR latices should have normally been coagulated, which was not observed.

6) Zeta potential measurements of tannin samples fell into a range where they should be coagulated, but they were fluid.

7) Tannin treatment with non-ionic surfactant generally gave better results of stability than the ionic type.

8) A tannin sample with non-ionic surfactant showed a higher dynamic viscosity pattern than its ionic counterpart.

9) Similar behavior is observed with the hydrodynamic particle diameter measurements; the non-ionic surfactant tannin treatment resulted in higher values than the tannin sample treated with ionic surfactant.

This whole set of objective facts based on laboratory results can be used to support the suggested model where tannin molecules complex with proteins surrounding the rubber particles in NRL, building a kind of shield which, based on steric hindrance, hinders the particles from coalescing to each other. This condition would keep the colloid system fluid and in a good state, in contrast with the normally observed particle protection acting through ionic forces. Water molecules attracted to tannin hydroxyls on the opposite side of particles would bring still more strength to the shield protection.

As proposed and observed, this shield model illustrates that field latex was stabilized against coalescence without using the traditional preservative ammonia. Therefore, the same shield built around the particles would be hindering them from merging to each other, as well as avoiding the bacterial attack to the proteins surrounding the rubber particles, which is associated with the latex decay.

Moreover, if this model is acceptable to explain the obtained results, it could be expected that proteins linked with tannin molecules, being part of the shield themselves, would not act as allergens. Because of experimental limitations at the Chemistry Institute of the University of Brasilia, this result was not totally proven from the experiments, as was the NRL protection against decay and coalescence without ammonia. The full picture of allergenic decrease resulting from tannin treatment will be better supported with specific experimental procedures, including proper methodology and pilot production of gloves for practical testing. However, the signs which were possible to get in this experiment from the ELISA are much in line with the rationale that tannin is really preventing the proteins from reacting as normal proteins, including the allergenic reactions, by surrounding the particles and building the shield, or by stabilizing the latex serum, in general.

Conclusion

This research work investigated the use of vegetable tannin to produce low protein latex with decreased allergenic action. At the same time, tannin action through protein complexation provided an alternative route to preserve latex against bacterial decay and physical coalescence without ammonia. Although the proposal is sound in technical and scientific backgrounds, trials of this sort were not found in the scientific literature.

The main conclusion of this study is the importance of introducing tannin as a new and potential component of hevea latex chemistry research. In order to become a protagonist with consistent contribution, it depends on further studies in order to define the sequence of reagents mixture, the general processing system and other aspects. However, there are steps between the bench scale and the field reality that demand much more research work to find the proper formulations and process parameters.

The answer to the initial question on the efficacy of using tannin to preserve the NR latex against decay was found and is positive. The possibility of having ammonia-free latex without expensive additives and simple processing is already and per se an interesting finding. If, in addition to this, the possibility can be confirmed of getting rid of latex allergens with very simple technical processing, then this research will be converted to a significant contribution to the latex industry.

This article is based on a paper presented at the 192nd Technical Meeting of the Rubber Division, ACS, October 2017.

References

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(2.) M.A. Perez, R. Rengifo, C. Pereira and V. Hernandez, "Dividivi tannins: An ecological product for water-based drilling fluids, " Environment, Development and Sustainability (2016), pp. 1-15, DOI: 10.1007/SI0668-016-9829-0.

(3.) J.B. Heredia and J.S. Martin, "Removing heavy metals from polluted surface water with a tannin-based flocculant agent, " Journal of Hazardous Materials (2009), 165 (1-3), pp. 1, 215-1, 218, DOI: 10.1016/J.JHAZMAT.2008.09.104.

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(9.) O. Chaikumpollert, et al., "Preparation and characterization of protein-free natural rubber, " Polymers for Advanced Technologies (2012), Vol. 23, No. 4, pp. 825-828, DOI 10.1007/ S00396-011-2549-Y.

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by Floriano Pastore, Jr., Joao Bosco Peres, Jr., Julia Kramer, Natalia Gomes and Leonardo Patemo, University of Brasilia, and Carlos Wolf, Tanac S.A., Montenegro, RS, Brazil

Caption: Figure 1--tannin molecular structure (ref. 8)

Caption: Figure 2--interaction model between tannin (at the top) and collagen proteins (at the bottom) (ref. 8)

Caption: Figure 3--a schematic representation of the shield model for the action of tannin on the latex colloid stability based on steric hindrance

Caption: Figure 4--pH results: LA (low ammonia), LAB (low ammonia and bactericide), HA (high ammonia), TBL (tannin, bactericide and ionic surfactant) and TBR (tannin, bactericide and non-ionic surfactant)

Caption: Figure 5--zeta potential (mV) of samples as a function of time
Table 1--treatments and formulations

Sample                                 Meaning and function

LA                                 Control with low ammonia

LAB                Control with low ammonia and bactericide

HA                                Control with high ammonia

TBL            Treatment with tannin, bactericide and ionic
                                           surfactant, pH 8

TBR        Treatment with tannin, bactericide and non-ionic
                                            surfactant pH 8

Table 2--results of smell test

                                  Smell score
                                  Time (hours)

Sample         6    30         54     78    150    222    318    408

LA            10     6    Coa (a)    Coa    Coa    Coa    Coa    Coa
LAB       Am (b)    Am         Am     Am     Am     Am      5      4
HA            Am    Am         Am     Am     Am     Am      3      2
TBL           10     7          7      7      7      6      4      5
TBR           10    10         10      9      9      8      5      8

(a) Coa means the sample has coagulated.

(b) Am means a strong ammonia smell hindered the test.

Table 3--quantification of allergenic proteins, Hev b5
and Hev b13, by TARRC/U.K.

          No centrifugation           One centrifugation
          (NC)                        (C1)

Sample        Hev b5       Hev b13        Hev b5       Hev b13

HA        [micro]g/g    [micro]g/g    [micro]g/g    [micro]g/g
               0.165           800         0.079           100
TBL            <0.02             -         <0.02             -
TBR            <0.02         1,600         0.073           120

          Double centrifugation
          (C2)

Sample        Hev b5       Hev b13

HA        [micro]g/g    [micro]g/g
               0.066            50
TBL                -             -
TBR            0.078            17
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Author:Pastore, Floriano, Jr.; Peres, Joao Bosco, Jr.; Kramer, Julia; Gomes, Natalia; Paterno, Leonardo; Wo
Publication:Rubber World
Date:Nov 1, 2017
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