Two new nontoxic, non-pathogenic strains of Sphingomonas elodea for gellan gum production
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.
References (10)
- et al.
Sphingomonas paucimobilis bacteremia in humans: 16 case reports and a literature review
J. Microbiol. Immunol. Infect.
(2010) - et al.
An insight into the emerging exopolysaccharide gellan gum as a novel polymer
Carbohydr. Polym.
(2013) - et al.
The genus Sphingomonas: physiology and ecology
Curr. Opin. Biotechnol.
(1996) CFR-code of Federal Regulations Title 21-21CFR172.665 Gellan Gum
(2015)- et al.
Occurrence, production, and applications of gellan: current state and perspectives
Appl. Microbiol. Biotechnol.
(2008)