Sterilization treatments on polysaccharides: Effects and side effects on pectin

https://doi.org/10.1016/j.foodhyd.2012.09.017Get rights and content

Abstract

Thermal and irradiation treatments represent techniques used for a wide range of materials intended for different applications in the food and medical industry aimed to decontamination and sterilization of different technologic processes. Traditional treatments with vapor combined with pressure, reactive gases, and radiation can be used for sterilization of medical implants and to prevent food deterioration. On the other side, these treatments can be employed to obtain a controlled distribution of molecular weight of polymers, as reported by literature.

Pectin, as many polysaccharides, may be susceptible to side effects and modifications caused by the sterilization treatments. In this perspective, physico-chemical and biological properties of pectin powders and solutions were investigated after sterilization. Traditional methods, i.e., ethylene oxide, gamma irradiation and moist heat sterilization, were compared and their effects on pectin structure were evaluated. Results indicate that each sterilization procedure affected pectin powders and solutions, resulting in a decrease of viscosity, molecular weight and rheological properties with respect of non-sterilized samples. For sterilized powders properties were better retained than for sterilized solutions, being EtO the optimal method for pectin powders and gamma rays, especially at low doses, for pectin solutions, independently of their structure and initial degree of esterification. Moist heat sterilization was found to significantly depolymerize pectin chains in solution, with the production of cytotoxic residues.

Highlights

► EtO, gamma rays and moist heat were considered to sterilize pectin powders and solutions. ► Pectin molecular weight and degree of esterification were modified by sterilization. ► EtO and irradiation minimized the side effects (degradation and cytotoxicity). ► Heating without additives or anti-oxidants resulted in the production of toxic compounds.

Introduction

Sterilization refers to the intense processes inactivating spores resulting in products that are shelf stable. It is usually achieved by applying heat, chemicals, irradiation, pressure and filtration (Mendes, Brandão, & Silva, 2007). Sterilization, however, may affect the surface or bulk properties of the sterilized material, inducing chemical or physical modifications that may alter the characteristics of the biomedical devices in terms of physico-chemical, morphological, mechanical and biological behavior.

Polymer solutions used in biomedical applications are in some cases sterilized by filtration, which physically separates the microorganisms from the polymeric solutions through filters with adequate pore sizes (usually 0.2 μm). Such process does not induce any modification of the polymeric structure (Schwan et al., 1986). However, this method is time consuming, difficult to perform on viscous solutions, and it should be always conducted in a sterile environment, with limitations when applied at industrial scale productions.

Other traditional methods of sterilization (i.e., ethylene oxide, irradiation, heat) are particularly damaging for polymers (Vink et al., 1986; Williams, 1997), also due to the similarity of the molecules constituting the polymeric chains, especially the natural-based ones, with the vital components of pathogens. As an example, ethylene oxide reacts with the amine groups of the microorganisms, denaturing their proteins, but it reacts also with the nucleophilic N-groups of polymers, such as chitosan, causing chain depolymerization (Politzer, Daiker, Estes, & Baughman, 1978).

In addition, natural polymers are susceptible to high temperatures and to the use of ionizing radiations, which may induce depolymerization, oxidation or the formation of free radicals (Leo, McLoughlin, & Malone, 1990), thus affecting the properties of the gels they may form, their mechanical strength, swelling and stability behavior (Munarin, Tanzi, & Petrini, 2012; Munarin, Petrini, Farè, & Tanzi, 2010).

Numerous studies have focused on the modifications of polysaccharides induced by sterilization, with the aim to optimize the sterilization conditions for different polysaccharides. The effects of sterilization depend upon the initial polymeric concentration, pH, and on the presence of water and oxygen. Wasikiewicz et al. (Wasikiewicz, Yoshii, Nagasawa, Wach, & Mitomo, 2005) focused on the effect of polymer concentration on their molecular and functional properties during sterilization: they found that the degradation of alginate and chitosan solutions sterilized with gamma rays is decreasing when increasing the polymer concentration. On the contrary, moist heat sterilization was found to be more damaging for highly concentrated solutions (Leo et al., 1990).

Other studies (Jacobs, 1985; Kang, Jo, Lee, Kwon, & Byun, 2005) evaluated more in detail the effects caused by the presence of oxygen during radiation sterilization of oligosaccharides. It was found that the breaking of C–C bonds decreased when irradiating the oligosaccharides in the absence of oxygen.

The effects of the initial pH of the polymer solutions during moist heat sterilization were studied by Daigle et al. (Daigle et al., 1997), who proved that moist heat sterilization of alginate is strictly dependent on the pH of solution, and that less depolymerization was produced if the solutions were buffered at pH 7–8 instead of the native, acidic pH (pH 6).

Due to the unique physico-chemical and biological properties, polysaccharides are employed in various fields, such as food processing, agriculture, wastewater purification, pharmaceutical, paper and textile industries, photographic processing as well as biomedical applications (i.e., encapsulation matrix for cells, genes and enzymes, wound dressing materials) (Blackburn, 2004; Colegrove, 1983; Mishra et al., 2005; Munarin, Petrini, Bozzini, & Tanzi, 2012, Munarin et al., 2011; Soma, Williams, & Martin, 2009; Song, Dong, Ragauskas, & Deng, 2009; Shibata et al., 1993). The determination of the mechanisms involved during sterilization of polysaccharides is therefore a fundamental step toward their industrial exploitation.

Among polysaccharides, a lack of information on the effects of sterilization on pectin is noticeable. The aim of the present work is to evaluate different sterilization techniques so as to indicate which ones are minimally affecting the properties of pectin powders and solutions. Sterilization was performed on pectins with different degree of esterification, polymeric concentration and water content. The research rationale is represented in the scheme of Fig. 1.

Section snippets

Materials and methods

Two pectin batches with different degree of esterification and molecular weight (Table 1) were analyzed in this work before and after sterilization. LM (pectin CU701) was kindly provided by Herbstreith & Fox (Neuemberg, Germany), while HM was purchased from Sigma Aldrich.

Intrinsic viscosity and molecular weight of sterilized pectin samples

All the considered sterilization techniques had significant effects on the intrinsic viscosity (Figs. 2 and 3) for LM and HM pectins. The effect of radiation doses for gamma ray sterilization consisted in a significant reduction of the intrinsic viscosity value when using the traditional sterilization dose (25 kGy) for both LM and HM (Fig. 2 and Table 2). Moderate variations in intrinsic viscosity and molecular weight were observed at lower sterilization doses (6 and 15 kGy).

From GPC analyses (

Ethylene oxide sterilization

Ethylene oxide is generally used to sterilize temperature-sensitive devices, such as natural polymers, whose properties (i.e., viscosity, molecular weight and mechanical properties) are dramatically lowered at high temperatures. The destruction of pathogen agents occurs through the alkylation of nitrogen groups and phosphoric acid esters of DNA (Hsiao, Liu, Wen-Neng Ueng, & Chan, 2012). However, the alkylation may also affect the properties of polyuronates, adding –CH2–CH2–OH groups in place of

Conclusions

The sterilization techniques considered in this work deeply affected the molecular weight and, at a lower extent, the degree of esterification (DE) of pectin samples. Physico-chemical characterization and cytotoxicity studies demonstrated the suitability of EtO and gamma rays sterilization as procedures to sterilize pectin powders and solutions, whereas the moist heat treatment seems not appropriate for pectins because of the side effects produced by heating. Therefore, the use of additives or

Acknowledgments

This work was supported by grants from Fondazione Cassa di Risparmio di Trento e Rovereto within the project “Biocompositi a base di polisaccaridi e calcio fosfati come riempitivi per tessuti molli” to M.C. Tanzi.

The authors wish to acknowledge Herbstreith & Fox (Neuenburg, Germany) for kindly providing the LM pectin used in this work and Eurocoating (Pergine, TN, Italy), for the sterilization of pectin powders and solutions.

The authors are also thankful to Monica Moscatelli for her technical

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