Enzyme resistance and biostability of hydroxyalkylated cellulose and galactomannan as thickeners in waterborne paints

d te ia, Accepted 13 June 2011 Available online 13 September 2011 Keywords: derivatives employed as emulsion stabilizers, thickeners, rheology modifiers, suspending or conditioning agents in a wide range of fields, such as oil well drilling, food and feed, personal care, detergents, textile printing, building materials, paints and coatings, and paper manufacturing (Risica et al., 2005). average ratio of mannose to galactose of 1.6e1.8 to 1 (McCleary et al., 1985). Cellulose is the primary product of photosynthesis in plants and is the most abundant renewable polymer on the biosphere. It is a linear polymer composed of units of D-glucopyranose linked by b-1,4 glycosidic bonds (Zhang et al., 2006), in which the anhydrous cellobiose represents the repeating unit (Zhang and Lynd, 2004). Hydroxyalkylation, performed with ethylene or propylene oxide, is the most commonly applied reaction on both polysaccharides to * Corresponding author. Tel.: þ39 0331 715464; fax: þ39 0331 715205. Contents lists available at International Biodeterior .e International Biodeterioration & Biodegradation 69 (2012) 106e112 E-mail address: [email protected] (Y.M. Galante). Polysaccharides of plant and microbial origin are increasingly applied in a growing number of fields, either in their natural form or following chemical/biochemical modifications. They are non- fossil raw materials from renewable sources and often represent an excellent alternative to traditional chemical polymers obtained from oil-derived monomers, such as polyacrylates. Indeed, the production/modification processes of polysaccharides conform largely to the Twelve Principles of Green Chemistry (Anastas and Warner, 1998). Second only to starch, cellulose and gal- actomannan are commonly used to produce a wide range of endosperms of a shrub (Cyamopsis tetragonolobus) commonly referred to as guar, grown predominantly in the northwestern areas of India and southeast Pakistan (Kay, 1979). Guar gum (GG) is obtained by thermo-mechanical, solvent-free processing of seeds with no generation of byproducts or environmental pollution. It is estimated that some 200,000e250,000 tons of guar gum are produced per year, from about 700,000 tons of seeds (Ashraf et al., 2005). The polymeric structure of galactomannan is composed of a backbone of mannose units linked by b-1,4 glycosidic bonds and side units of galactose linked by a-1,6 glycosidic bonds, with an Polysaccharide hydroxyalkylation Hydroxyethyl cellulose Hydroxypropyl guar Biostability Enzyme resistance Waterborne paints 1. Introduction 0964-8305/$ e see front matter � 2011 Elsevier Ltd. doi:10.1016/j.ibiod.2011.06.009 one of the most common chemical reactions applied to modulate the rheological profile and other properties of polysaccharides. Hydroxyethyl cellulose (HEC) and hydroxypropyl guar (HPG) are widely used as thickening and stability agents in waterborne paints. Hydroxyalkylation also increases the resistance of polysaccharides to enzyme degradation due to steric hindrance by the substituents on the susceptible bonds along the polysaccharide backbone. This feature of “enzyme resistance” is often referred to as “biostability,” yet it does not mimic a real-life situation of microbial contamination that can occur in a production plant or storehouse. We have compared viscosity decreases of HECs and HPGs in the presence of their specific hydrolyzing enzyme (cellulase and mannanase, respectively) to actual microbial contamination by consortia of fungi or bacteria. We found that the behaviour of HEC and HPG solutions inoculated with microorganisms differs and cannot be predicted from enzyme challenge data alone. Thus, “enzyme resistance” and “biostability” are not equivalent properties. For practical purposes, it is important to bear this in mind when selecting the most appropriate polysaccharide thickener, the manufacturing conditions of waterborne paints and the optimal stage of biocide addition. � 2011 Elsevier Ltd. All rights reserved. Galattomannan is a branched polysaccharide found in the seed Received in revised form 13 June 2011 manufacturing, and oil operations. Hydroxyalkylation, performed with ethylene or propylene oxide, is Article history: Received 11 January 2011 Chemically modified polysaccharides are widely used as rheology modifiers in several applications, such as food and feed, personal care, detergents, textile printing, building materials, paints and coating, paper Enzyme resistance and biostability of hy and galactomannan as thickeners in wa S. Cheronia, B. Gattib, G. Margheritisb, C. Formantic a Laboratory of Biotechnology, Lamberti S.p.A., Via Piave 18, 21041 Albizzate (VA), Italy bConstruction Laboratory, Lamberti S.p.A., Via Piave 18, 21041 Albizzate (VA), Italy a r t i c l e i n f o a b s t r a c t journal homepage: www All rights reserved. roxyalkylated cellulose rborne paints L. Perroneb, Y.M. Galantea,* SciVerse ScienceDirect ation & Biodegradation lsevier .com/locate/ ibiod Experimentally, the concept of “biostability” is inappropriately applied to the behaviour (i.e., extent of viscosity decrease) of an HEC or HPG solution upon incubation with a specific enzyme (i.e., cellulase or mannanase, respectively) as a function of time under standard conditions. Because it does not truly represent a microbial contamination that can occur in the laboratory, production plant, or construction site, we believe that a more appropriate definition of this property would be “enzyme resis- tance.” Results of actually challenging a thickener solution with microbial inocula should provide more accurate information and sound evidence. To our knowledge, no published investigation has reported on a comparison of enzymatic and microbial challenges on the viscosity of solutions of HEC and HPG. We have investigated this issue and compared the stability of property of “biostability,” as indicated by the manufacturer, is ration & Biodegradation 69 (2012) 106e112 107 modify their solubility and viscoelastic properties (Lapasin and Pricl, 1995; Nussinovitch, 1997; Cheng et al., 2002a). Hydroxyethyl cellulose (HEC) is obtained by reacting ethylene oxide with finely milled cellulose (from wood pulp or cotton linters) in a hydro-alcoholic medium with stoichiometric amounts of alkali; this is followed by purification, drying, milling, and particle sizing (Davidson, 1980). One of the most common uses of HEC is in waterborne paint formulations, as a thickening, associa- tive, water retention, and film-forming agent (Tothill and Seal, 1993; Sau, 1999; Calbo, 1992). Hydroxypropyl guar (HPG) is a more recent development in the field of polysaccharide derivatives and is produced by reacting GG with propylene oxide in the presence of catalytic alkali in a hydro- alcoholic medium (Cheng et al., 2002b). HECs and HPGs share several industrial applications and are structurally characterized by their degree of molar substitution (MS), which represents the average number of hydroxyalkyl groups linked to each monosaccharide unit (Cheng et al., 2002a,b). The hydroxyalkylated polysaccharides must have defined values of MS in order to make them suitable for the paint industry. Commonly used thickeners have MS values in a range going from 2.3. Enzymatic challenge tests The HECs or HPGs were dissolved at 2% (w/w) in water, pH was adjusted to 6.0 and 7.0, respectively, with phosphate buffer plus 0.02% sodium azide added as preservative. Those pH values were chosen because the carbohydrolases used in these experiments (cellulase andmannanase) have acidic to near neutral pH optima of activity. Two units of Indiage Max L were added to 350 g of an HEC solution and 5 units of Gamanase 1.0 L to 350 g of an HPG solution. The mixtures were incubated at room temperature (20e23 �C) under constant stirring. Enzymatic challenges of the waterborne paints of Fig. 3 were performed as follows: 350 g of each paint, formulated with a different HPG, were supplemented with 3.5 units of NS 44076 (an alkaline galactomannanase, because the pH of the paint was adjusted to 8.5e9.0) and all samples were incubated as described above. Viscosity was measured at various time intervals with a Brook- � ration & Biodegradation 69 (2012) 106e112 (z2.5e3.0), high (z3.0e3.5), and very high (3.5e4.0), and for HPG as low (z0.6), medium (z0.8e1.0), high (z1.2e1.5), and very high (>1.5). The fungal consortium used was composed of the following strains: Aspergillus oryzae (DSM 12635), Cladosporium cladospor- ioides (IMI 178517), Geotricum candidum (DSM 12636), Paecilomyces variotii (IMI 114930), and Penicillium ochrocloron (IMI 061271). This consortiumwas further boosted with a strain of Trichoderma viride, a strong carbohydrolases fungal producer (Kubicek and Harman, 1998), kindly supplied by Thor Italy. (DSM ¼ Deutsche Sammlung von Mikroorganismen; IMI ¼ International Mycological Institute, Egham, UK [CABI Bioscience].) The bacterial consortium used was composed as follows: Aero- monas hydrophila (NCIMB 9233), Alcaligenes faecalis (NCIMB 13145, IBRG P21), Cellulomonas flavigena (NCIMB 8073), Corynebacterium ammoniagenes (NCIMB 8143), Enterobacter aerogenes (NCIMB 10102), Escherichia coli (NCIMB 8114), Klebsiella pneumoniae (NCIMB 8021), Proteus vulgaris (NCIMB 4175), Providencia rettgeri (NCIMB 10842), Pseudomonas aeruginosa (NCIMB 8295), Pseudo- monas stutzeri (NCIMB 11359), and Serratia liquefaciens (NCIMB 12815), also from Thor Italy. (NCIMB ¼ National Collection of Industrial and Marine Bacteria.) Microbial challenges were carried out following the IBRG (International Biodeterioration Research Group, UK) guidelines (IBRG, 2000; see also: www.ibrg.org), by adjusting the total cell count concentration inoculated in the test solutions. The cellulase used in the enzymatic challenge test of HEC was Indiage Max L from Genencor-Danisco, which is a genetically modified cellulase from Trichoderma reesei, depleted in exogluca- nase and enriched in endoglucanase activities, with an optimal activity at pH 4.5e6.0 (Galante and Formantici, 2003). The mannanases used in the enzyme challenge experiments were Gamanase 1.0 L, a food-grade, non-recombinant enzyme Table 2 Features of hydroxypropyl guars (Esacol� HPG 1 to 5 and HPG-Lucid) studied. HPG sample MS (hydroxypropyl group) Viscosity (2% solution at pH 7, 20 �C, 20 rpm) mPa*s Esacol� HPG-1 >1.5 7200 � 400 Very high Esacol� HPG-2 z1.2e1.5 8900 � 450 High Esacol� HPG-3 z1.2e1.5 8100 � 400 High Esacol� HPG-4 z0.8e1.0 12,300 � 600 Medium Esacol� HPG-5 z0.8e1.0 21,400 � 1000 Medium HPG-Lucid z0.6 14,200 � 700 Low S. Cheroni et al. / International Biodeterio108 active at a slightly acidic to neutral pH; and NS 44076, an alkaline galactomannanase active at pH 8.0e9.0; both are from Novozymes. 2.2. Characterization of carbohydrolases Protein concentration was determined by the Lowry method (Lowry et al., 1951). Cellulase and mannanase activities were determined in aqueous buffer by the Cellazyme or the Beta- Mannazyme methods (T-CCZ200 03/06 and T-MNZ 03/06, respec- tively, from Megazyme), with either azurine dyed, crosslinked insoluble hydroxyethyl cellulose or azurine dyed, crosslinked carob galactomannans, respectively, as substrate, both in the form of water-insoluble tablets (www.megazyme.com). With these methods, one unit of enzyme activity (U) is defined as the amount of enzyme that determines an increase in absorbance of 1 unit at a wavelength of 590 nm under the specified conditions over a 10- min reaction time. field SynchroeLectric Viscometer at 20e23 C and 20 rpm using the following spindles according to the corresponding viscosity intervals: spindle 2 for 500e1400 mPa*s, spindle 3 for 1500e3500 mPa*s, spindle 4 for 3600e6500 mPa*s, spindle 5 for 6600e14,000 mPa*s, and spindle 6 for 14,100e40,000 mPa*s. With both HEC and HPG, viscosity values were recorded after a 1 min spindle rotation to equilibrate the system. 2.4. Composition of waterborne paints The composition of thewaterborne paints used in the enzymatic challenge tests of Fig. 3 is reported in Table 3. 2.5. Microbial challenge tests Eight hundred grams of a 2% solution was prepared with all samples of HEC or HPG in MilliQ water. The pH was adjusted to 8.5e9.0 with ammonia. Each solution was divided into two 400-g aliquots, one of which was inoculated with the appropriate microbial consortium, while the other was kept as a reference blank. All material (MilliQ water, glassware, etc.) was autoclaved at 121 �C for 20 min to ensure sterility. To 400 ml of HPG or HEC solution, the fungal or bacterial consortium (IBRG, 2000; see also: www.ibrg.org), was inoculated to a final count of 105 CFU g�1. After completemixing of the solution, it was incubated at 30 �C for the duration of the experiment. Fig. 1. Enzymatic hydrolysis of HEC: Brookfield viscosity profile versus time of three commercial samples challenged with cellulase Indiage Max L at pH 6.0. Natrosol 250 HR (A), Natrosol 250 HBR (þ), Natrosol Plus 330 (�). 4% glucose agar (65 g l of Fluka 84088) was used as culture medium, while tryptic soy agar (40 g l�1 of Sigma, T4536) was used Fig. 2. Enzymatic hydrolysis of HPG with increasing MS: Brookfield viscosity profile versus time of HPGs challenged with mannanase Gamanase at pH 7.0. HPG-2 (A), S. Cheroni et al. / International Biodeterioratio for bacterial counts (European Pharmacopoeia, 2009). Viscosity was measured under a vertical laminar flow sterile hood at various time intervals with a Brookfield SynchroeLectric Viscometer at 30 �C and 2.5 rpm using the following spindles according to the corresponding viscosity intervals: spindle 2 for 3300e11,500 mPa*s, spindle 3 for 11,600e28,000 mPa*s, spindle 4 for 28,100e52,000mPa*s, and spindle 5 for 52,100e104,000mPa*s. With both HEC and HPG, viscosity values were recorded after a 1 min of spindle rotation to equilibrate the system. 3. Results and discussion 3.1. Enzymatic hydrolysis of commercial HEC and HPG with increasing MS To determine the total microbial count, aliquots of HEC or HPG were diluted as appropriate in tryptic soy broth (30 g l�1 of Sigma, T8907) to reach a cell concentration that could be easily counted in a petri dish, following plating of 1ml of the dilutedmixture on solid agar and incubation at 30 �C for 72 h. For fungal counts, Saboraud �1 HPG-4 (:), HPG-5 (-), Lucid (C). Etherification of polysaccharides, such as hydroxyalkylation, improves their resistance to enzyme hydrolysis due to steric Fig. 3. Enzymatic hydrolysis of waterborne paints thickened with HPG. Brookfield viscosity profile versus time of paints challenged with an alkaline mannanase at pH 8.5e9.0. HPG-1 (C), HPG-3 (A), HPG-5 (-). hindrance of the substituents with the susceptible glycosidic bonds along the polysaccharide backbone (Glass and Lowther, 1978). Three types of available commercial HECs were selected according to their increasing MS: medium, high, and very high (see section on materials, and Table 1). Fig. 1 shows their Brookfield viscosity profile over time when challenged at pH 6.0 with Indiage Max L, a cellulase complex enriched in endo-cellulase activity and active at acidic pH. The claim of the manufacturer e that the so- called “bioresistant” HECs (i.e., Natrosol 250 HBR and Natrosol Plus 330) are much more resistant to degradation by a specific hydrolytic enzyme than their counterpart not considered “bio- resistant” (i.e., Natrosol 250 HR) ewas fully confirmed. Indeed, the latter quickly lost viscosity upon cellulase addition, following the onset of depolymerization. We assume that the significantly lower susceptibility to enzyme degradation of Natrosol 250 HBR and Natrosol Plus 330 versus Natrosol 250 HR is due to their higher MS and very likely also to a more uniform distribution of ether substituents along the polymer backbone. The “very high MS” Natrosol HBR (MS 3.5e4.0) turned out to be, as expected, the most resistant to cellulase hydrolysis. A comparable patternwas obtained when six HPG samples with increasing MS were challenged with a mannanase, as shown in Fig. 2, which reports the viscosity decrease of water solutions of HPG at pH 7.0, supplemented with Gamanase 1.0 L, a food-grade, non-recombinant mannanase, active at slightly acidic to neutral pH. From these results, it can be concluded that the rate of poly- Table 3 Composition of thewaterborne paints used in the enzymatic challenge tests of Fig. 3. Component Function % w/w Calcium carbonate Extender 50 Acrylic ester-styrene copolymer Binder 10.0 Polycarboxylic acid (Na salt) Dispersing agent 0.3 Defoaming agent 0.1 White spirit Coalescing agent 1.0 Titanium dioxide Whitening agent 5.0 HPG xa Esametaphosphate sodium salt Sequestrant 1.0 Biocide 0.05 Ammonia 25% To adjust pH at 8.5e9.0 Water To reach 100 a The following amounts of HPG were added in order to reach a final viscosity of 20,000 mPa s (at 20 rpm and 20 �C): HPG-4, 1.08%; HPG-5, 0.76%; HPG-1, 1.2%. n & Biodegradation 69 (2012) 106e112 109 saccharide hydrolysis, as estimated by the decrease in Brookfield viscosity, appears to be inversely related to the degree of MS of HPG. It is obvious from Fig. 2 that while straight GG exhibits an extremely rapid fall in viscosity (t1/2 < 0.5 h), roughly equivalent to an HPG with low substitution, the HPGs with high MS (i.e., 1.2e1.5 or higher) have a t1/2 of 4 h and over, with a bimodal curve of viscosity decrease which, after a rapid fall, levels off or slowly decreases. A similar set of experiments was performed on a complete mixture of a standard waterborne paint formulation at pH 8.5e9.0, thickened with three HPGs with increasing MS (see Table 3), challenged with an alkaline galactomannanase (NS44076 from Novozymes) in order to assess whether the same pattern would be found as in water solution. This particular enzyme was chosen because of its alkaline pH optimum, which is similar to the one of the paint formulation chosen. Once again, the higher the MS of the HPG used as thickener, the slower was the decrease in viscosity and the lesser the extent of viscosity fall caused by enzyme action (see Fig. 3). These results also confirmed that, in the case of a typical waterborne paint formulation for wall painting, a high or very high molar substitution of GG provides significantly better “enzyme resistance” to the polysaccharide derivative. It should be mentioned that the HPG-Lucid sample was not considered in this or further experiments because it was found to contain a high concentration of isothiazolinones as protective biocides that would have hampered the microbial challenge tests (see below). 3.2. Fungal and bacterial challenge of HEC and HPG It was mentioned in the introduction that the foregoing results of susceptibility of either HEC or HPG to enzymatic degradation in aqueous solutions brought about by a specific enzyme (cellulase or mannanase, respectively) can be questioned as not being truly representative of a real-life situation, where the accidental pres- ence of microorganisms, rather than of “free” enzymes, is a rather with a higher or lower cell count) did not change the overall pattern, but only managed to slow down or to accelerate the rate of viscosity decline. Samples of HEC or HPG not inoculated and incubated in parallel showed no loss of viscosity or cell growth. While it can be argued that these results can be explained by the fact that the inoculated microbial species produced little, if any, mannanase compared to cellulase, it should be emphasized that the microbial consortium used in our experiments is the one developed Fig. 6. Challenge tests of HEC and HPG solutions with the bacterial consortium at pH S. Cheroni et al. / International Biodeterioration & Biodegradation 69 (2012) 106e112110 common event. In a paint production plant or storehouse, deteri- oration of the thickener or paint stock solution can quite easily occur due to microbial contamination, prior to addition of an appropriate biocide to the final product during in-can packaging or also in the presence of such biocide. Indeed, the hydrolytic action of secretedmicrobial exo-enzymes can proceed even in the absence of living cells, if a previous microbial contamination has occurred somewhere upstream in the production process. Thus, we have studied the effect of inoculating a complex, well- defined microbial consortium into HEC and HPG solutions and monitored two crucial parameters over time, namely Brookfield viscosity and total microbial count. As far as we are aware, this approach (i.e., challenge with live microorganisms) to character- izing the stability of polysaccharide derivatives used as paint thickeners has not been previously reported by others, while several reports of enzyme challenge have been published. Accordingly, 2% (w/w) solutions of HEC or HPG at pH 9.0 were inoculated to a final count of 105 CFU ml�1 with a fungal consor- tium, developed by the IBRG in the year 2000 (www.ibrg.org) for challenge tests of waterborne paints and supplemented by a strain of T. viride, a strong fungal carbohydrolase producer (Kubicek and Harman, 1998), and incubated at 30 �C. Fig. 4 shows viscosity decrease versus time of HEC and HPG, while Fig. 5 shows the total microbial count determined in all hydroxyalkylated polysaccharide solutions, expressed as colony- forming units per gram of mixture (CFU g�1). In three separate cycles of fungal inocula and prolonged incu- bation, the solutions of HEC, whether or not they were considered “enzyme resistant” (from Table 1 and Fig. 1), rapidly lost viscosity, presumably due to extensive degradation of the cellulose backbone. On the other hand, the HPG solutions (from Table 2, Fig. 4. Challenge tests of HEC and HPG solutions with the fungal consortium at pH 9.0: Brookfield viscosity profile versus time. HPG-4 (A), HPG-5 (-), HPG-2 (:), Natrosol 250 MHBR (�), Natrosol 250 HBR (þ), Natrosol Plus 330 (�). Figs. 2 and 3) exhibited a much slower and more limited extent of viscosity loss compared to HEC. However, in all samples, a steady increase in fungal cell growth was observed over time (see Fig. 5). Considering the results of the enzymatic challenges (Figs. 2 and 3), no obvious correlation could be observed between viscosity loss and the degree of MS of the HEC and HPG studied. More surprisingly, the remarkable “enzyme resistance” property of HEC (see also Fig. 1) did not manifest itself as “bioresistance” in the presence of a fungal inoculum and ensuing cell growth. Indeed, all HEC studied rapidly lost viscosity, much more so than HPG, which, in contrast, appeared to be considerably more “biostable.” Varying cell concentration of the inoculum (i.e., challenging Fig. 5. Challenge tests of HEC and HPG solutions with the fungal consortium at pH 9.0: total microbial count versus time. HPG-4 (A), HPG-5 (-), HPG-2 (:), Natrosol 250 MHBR (�), Natrosol 250 HBR (þ), Natrosol Plus 330 (�). 9.0: Brookfield viscosity profile versus time. HPG-4 (A), HPG-5 (-), HPG-2 (:), Natrosol 250 MHBR (�), Natrosol 250 HBR (þ), Natrosol Plus 330 (�). Thus, “enzyme resistance” and “biostability” are not equivalent complex consortium of microbial species used in the laboratory for ratio and recommended by the International Biodeterioration Research Group, UK (IBRG, 2000; see also: www.ibrg.org) e further boosted by the addition of a strain of T. viride for challenge tests of water- borne paints. Thus, it is assumed to be representative of an actual accidental contamination by fungi. If we accept that the above approach more closely mimics a real-life situation, one can conclude that HPGs not only have equivalent “bioresistance” properties to HEC in the presence of an accidental environmental microbial contamination, but even higher. An analogous set of experiments was carried out with the bacterial consortium described in the methods section. The procedure was similar to the one used for the fungal inoculum, as recommended by the IBRG. Fig. 7. Challenge tests of HEC and HPG solutions with the bacterial consortium at pH 9.0: total microbial count versus time. HPG-4 (A), HPG-5 (-), HPG-2 (:), Natrosol 250 MHBR (�), Natrosol 250 HBR (þ), Natrosol Plus 330 (�). S. Cheroni et al. / International Biodeterio Both HEC and HPG maintained about 80% of their initial viscosity for up to 20 days after bacterial inoculum, but in this case the difference in the rate and extent of viscosity decline between the two groups of polysaccharide derivatives was much less pronounced than in the fungal challenge (see Fig. 6). Bacterial growth occurred similarly in all polysaccharide solutions tested, as shown in Fig. 7. The results of Fig. 6 appear to be in agreement with what was concluded by Tothill and Seal (1993), who pointed out that “bacteria are known to have a generally low yield of extracellular cellulases” (we would extend this statement to carbohydrolases in general) “and can cause a moderately steady decrease in paint viscosity.” Those authors, however, did not report an experiment of live bacterial challenge to support their conclusion. 4. Conclusions As we have argued in the introduction, using “biostability” to describe resistance of a polysaccharide derivative to enzymatic degradation can be misleading. We believe that a challenge per- formed with live microorganisms is a better approach. We have compared viscosity decrease of HEC and HPG solutions challenged with their specific hydrolyzing enzymes (cellulase and mannanase, respectively) to challenge experiments performed with microbial inocula of heterogeneous consortia of fungi or bacteria. We found that the behaviour of inoculated HEC and HPG challenge tests cannot entirely represent all possible microbiolog- ical contaminations that might occur in real life. Thus, we have relied on a mix of moulds and yeasts, recommended to be used in the performance of challenge tests to evaluate the degree of “biocidal protection” of waterborne paint formulations. To what degree the fungal consortium used in the present work, under the growth conditions of our experiments, produces cellulases, man- nanases, and other carbohydrolases, is very hard to assess. However, this feature has relative relevance if we assume that it does simulate an actual environmental microbial contamination. If so, this approach allows for a better evaluation of the stability of a polysaccharide thickener in solution or paint formulation. Therefore, the characteristic called “biostability” is a rather complex property of native and chemically modified macromole- cules, which should not be extrapolated exclusively from data of in- vitro “enzyme resistance.” Actual environmental contaminations are due to microorganisms, not to “free-floating” enzymes. We propose the following and more accurate definition of “biostability”: Biostability is the intrinsic property of a chemically modified polysaccharide to maintain its chemical structure and rheological properties in aqueous solutions in the presence of microbial contamination. It is not an absolute property, but a feature of increased stability of the modified biopolymer relative to its unmodified or limited modified form. Two main causes of paint viscosity loss (i.e., “thinning”) have been described: (1) microbial contamination, and (2) the disag- gregative effect of oxidizing and reducing agents (Tothill and Seal, 1993). Although the latter cannot be ruled out a priori, the major concern should always be to prevent microbial contamination as early as possible all along the production/storage chain. Chemical causes can easily be corrected by eliminating or deactivating the causative agent. From our own experience, as well as that of others who are active in several industrial fields, including paint, textile printing, leather finishing, ceramics, etc., various deteriorating conditions can frequently occur involving stock solutions of polysaccharide-based thickeners, rheology modifiers and of finished products. Therefore, proper operating conditions should be put in place as preventive measures in order to avoid microbial contaminations and viscosity decline, such as appropriate sani- tizing of water and facilities, followed by frequent monitoring of microbial count. 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Zhang, Y.H.P., Lynd, L.R., 2004. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnology and Bioengineering 88, 797e821. Zhang, Y.H.P., Cui, J., Lynd, L.R., Kuang, L.R., 2006. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 7, 644e648. S. Cheroni et al. / International Biodeterioration & Biodegradation 69 (2012) 106e112112 Enzyme resistance and biostability of hydroxyalkylated cellulose and galactomannan as thickeners in waterborne paints 1. Introduction 2. Materials and methods 2.1. Materials 2.2. Characterization of carbohydrolases 2.3. Enzymatic challenge tests 2.4. Composition of waterborne paints 2.5. Microbial challenge tests 3. Results and discussion 3.1. Enzymatic hydrolysis of commercial HEC and HPG with increasing MS 3.2. Fungal and bacterial challenge of HEC and HPG 4. Conclusions Acknowledgements References