Two new nontoxic, non-pathogenic strains of Sphingomonas elodea for gellan gum production

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Highlights

  • Two new Sphingomonas elodea strains (PHP1 and PBAD1) were developed for gellan production.

  • Nonpathogenic and nontoxic S. elodea strains must be used to produce gellan.

  • Studies were conducted to determine whether PHP1 and PBAD1 meet this requirement.

  • Results show that neither PHP1 nor PBAD1 are pathogenic or toxic.

Abstract

Two new strains of Sphingomonas elodea (designated as PHP1 and PBAD1) were tested for toxicity and pathogenicity in healthy Sprague-Dawley CD® IGS rats in separate studies. In each study, twelve rats/sex were administered ≥108 viable cells/rat by oral gavage, and four untreated rats/sex served as controls. Blood, feces, and selected organs/tissues collected at various times over the course of the 22 day study were evaluated for the presence of PHP1 or PBAD1 (depending on the study) by a validated method, to determine the potential for survival, propagation, or infectivity of PHP1 and PBAD1 cells in the rat. No mortalities, test substance-related changes in clinical or macroscopic findings, body weight or body weight gain were observed in treated animals compared with controls, indicating a lack of toxicity. PHP1 or PBAD1 were not detected in the tissue, fecal or fluid samples collected from treated animals. Therefore, neither PHP1 nor PBAD1 were pathogenic or acutely toxic under the conditions of the studies.

Introduction

Gellan gum (or simply, gellan), is a multifunctional gelling agent used in food, cosmetics, personal care products, pharmaceuticals and tissue culture media. Chemically, native gellan is a linear anionic polysaccharide with tetrasaccharide repeating units composed of two molecules of β-D-glucose, one molecule of L-rhamnose and one molecule of D-glucuronic acid, with two acyl substitutes (O-acetate and L-glycerate) linked to the same glucose residue (Fialho et al., 2008). The properties of gellan may be influenced by raw materials used in the synthesis of the gum, or by alterations in the pH, temperature and purification method of the gum (Fialho et al., 2008, Prajapati et al., 2013).

In the United States, the food additive regulation for gellan gum (21 CFR §172.665) specifically states that gellan must be produced by a non-pathogenic and nontoxic strain of Pseudomonas elodea (FDA, 2015), also known as Sphingomonas elodea (Vartak et al., 1995). Sphingomonas elodea is a gram-negative, non-motile, non-fermentative, aerobic bacteria characterized by an outer membrane that contains glycosphingolipids, but lacks lipopolysaccharide (White et al., 1996). Typically, gellan production by Sphingomonas elodea is a growth-associated process with a relatively low rate of sugar conversion (40–50%) (Fialho et al., 2008). Engineered strains of Sphingomonas elodea have been developed to increase substrate conversion and/or eliminate impurities that limit applications. PHP1 and PBAD1 block synthesis of polyhydroxybutyrate, an intracellular energy storage product (Wu et al., 2011). These strains also have modifications inactivating enzymes that limit use of the gellan gum in certain applications. Studies described in this manuscript were conducted to ascertain the potential for toxicity and pathogenicity of PHP1 and PBAD1.

Section snippets

Guidelines

The studies were conducted in accordance with Good Laboratory Practice (GLP), US FDA Toxicological Principles for the Safety Assessment of Food Ingredients, Redbook 2000, IV.C. 3 a: Short-Term Toxicity Studies with Rodents (2007) and U.S. EPA Health Effects Test Guidelines, OPPTS 885.3050, Acute Oral Toxicity/Pathogenicity (1996). Protocols for the studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the contract lab (Product Safety Labs, Dayton, New Jersey) prior

Validation studies

For PHP1, the microbial concentration of the inoculum used in samples collected from rat #460 (brain, liver, spleen, kidney, lymph nodes and blood) was 1.74 × 104 CFU/mL. The expected growth, assuming complete recovery, would be 174 CFU/plate for inoculated tissue and blood. For undiluted feces, approximately 35 CFU/plate was expected. The inoculum prepared for tissues collected from rat #493 (lung and feces only) contained approximately 1.91 × 104 CFU/mL, and was expected to produce

Discussion and conclusions

The food additive regulation for gellan gum (21 CFR §172.665) states that the gum must be produced by a nontoxic and non-pathogenic strain of Pseudomonas elodea (FDA, 2015). After the acceptance of the food additive petition by FDA, Pseudomonas elodea was placed into the genus Sphingomonas and is now generally recognized as Sphingomonas elodea (Vartak et al., 1995). Thus, for the genetically modified Sphingomonas elodea strains PHP1 or PBAD1 to be used in gellan gum production, the strains must

Conflicts of interest

All authors have a financial relationship with the sponsor of the studies and manuscript.

Acknowledgements

The authors thank Silvia Ulm and Lu Zhao for assistance in preparing the manuscript.

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