# Patent

## Title
Genetically purified gellan gum

## Abstract

Mutational inactivation of proteins involved in para-cresol production in certain milk products results in improved taste and odor. The undesirable para-cresol forms over time as a result of enzymes produced by the bacterium that produces gellan gum. Since the gellan is typically used in a relatively unpurified form, the enzymes are added to the milk along with the gellan. Inactivation of the enzymes is a genetic means of eliminating the enzymes without requiring any additional purification or processing.

## Background
This application claims priority under 35 U.S.C. 119(e) to provisional application 60/580,730 filed on Jun. 21, 2004.


A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The invention relates to the field of food additives. In particular it relates to the field of dairy food additives. More particularly it relates to additives to sterilized milk products.
BACKGROUND OF THE INVENTION
Gellan gum is an extracellular polysaccharide produced by the bacteria Sphingomonas elodea. Gellan gum produced by S. elodea is commercially available as Kelcogel LT100® from CP Kelco, San Diego, Calif. Commercially, gellan gum is formed by aerobic fermentation. Upon completion of fermentation, the broth is pasteurized to kill viable cells prior to recovery of the gum from the fermentation broth.
Gellan gum comprises the sugars glucose, glucuronic acid, and rhamnose in a 2:1:1 molar ratio, which are linked to form a tetrasaccharide repeat unit. Native gellan gum is acetylated and glycerylated on the same glucose residues. On average, there is one acetyl group and one half glyceryl group per tetrasaccharide repeat unit.
The method of recovery of the gellan gum affects the characteristics of the gum. Direct recovery yields a soft, flexible gel. Gellan gum has long been used in cultured, retorted, and frozen dairy products due to its textural and rheological properties. However, an off-flavor and odor develop in otherwise shelf-stable, milk-based, gellan-containing products; this flavor and odor render the foods unpalatable. The off-flavor and odor have been linked to the formation of para-cresol from substrates in milk, e.g., para-cresyl sulfate and para-cresyl glucouronide. Para-cresol is detectable in milk-based, gellan-containing products that have been treated at ultra high temperatures and stored at room temperature.
In an effort to eliminate this problem, gellan has been deacylated with hot alkali treatment. While effective in eliminating the para-cresol, the deacylation processing makes the gellan gum more brittle and less useful for certain food applications. Another approach to eliminate this problem is the pre-treatment of native gellan gum with a denaturing agent, such as sodium hypochlorite or potassium hydroxide. This approach adds material and processing costs. There is a need in the art for a gellan product which does not produce para-cresol upon prolonged storage in a sterilized dairy product and which does not require extra processing steps.
BRIEF SUMMARY OF THE INVENTION
In a first embodiment a composition is provided which comprises gellan gum substantially free of arylsulfatase protein.
In a second embodiment a composition is provided which comprises gellan gum substantially free of β-glucuronidase protein.
In a third embodiment of the invention a composition is provided which comprises gellan gum substantially free of both arylsulfatase and β-glucuronidase proteins.
In a fourth embodiment of the invention a method is provided for producing a gellan gum composition. Sphingomonas elodea is cultured in a culture medium. The Sphingomonas elodea produces no catalytically active arylsulfatase, or no catalytically active β-glucuronidase, or no catalytically active arylsulfatase and no catalytically active β-glucuronidase. The culture medium is collected. Gellan gum is precipitated from the culture medium.
A microbiologically pure culture of Sphingomonas elodea is provided in a fifth embodiment of the invention. It is arylsulfatase-deficient.
Another microbiologically pure culture of Sphingomonas elodea is provided in a sixth embodiment of the invention. It is β-glucuronidase-deficient.
Still another embodiment of the invention is a microbiologically pure culture of Sphingomonas elodea. It is deficient in both arylsulfatase and β-glucuronidase.
An eighth embodiment of the invention provides an isolated and purified polynucleotide encoding a Sphingomonas elodea arylsulfatase. The arylsulfatase has an amino acid sequence according to SEQ ID NO: 2.
A ninth embodiment of the invention provides an isolated and purified polynucleotide encoding a Sphingomonas elodea β-glucuronidase. The β-glucuronidase has an amino acid sequence according to SEQ ID NO: 5.
A tenth embodiment of the invention is an isolated and purified polynucleotide comprising Sphingomonas elodea genomic DNA. The genomic DNA comprises a deletion of all or part of its arylsulfatase coding sequence.
An eleventh embodiment of the invention is an isolated and purified polynucleotide comprising Sphingomonas elodea genomic DNA. The genomic DNA comprises a deletion of all or part of its β-glucuronidase coding sequence.
These and other embodiments of the invention as described in more detail below provide the art with cost-effective means to make a more consumer-acceptable, sterilized, gellan-containing, dairy product.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Genetic map of the genomic region around the arylsulfatase gene (atsA) and location of the regions amplified by PCR and cloned into plasmid pLO2. Plasmid pLO2 with the cloned PCR fragments was then used to replace this region of the genome with the deletion, by homologous recombination.
FIG. 2. Restriction map of the genomic region around the beta-glucuronidase gene (gusA) of Sphingomonas elodea. Positions of transposon insertions in clones BG-6 and BG-7 are indicated at the bottom.



DETAILED DESCRIPTION OF THE INVENTION
The present inventors have found that if either of the genes encoding the enzymes arylsulfatase and β-glucuronidase or both genes are mutationally inactivated in the bacterium Sphingomonas elodea, the bacterium produces a gellan that has superior properties for certain purposes. In particular, the gellan that is produced by such mutants imparts to sterilized milk products a longer shelf-life.
If one or both of the enzymes are not inactivated then they produce para-cresol from substrates (p-cresyl-sulfate and p-cresyl-glucuronide) found in the milk. The para-cresol imparts an odor and flavor that is generally unpalatable to consumers. Eliminating these enzymes reduces the rate at which para-cresol is produced in the sterilized milk or milk product on the shelf.
Mutations in either or both of the enzymes may be used to reduce the rate of para-cresol production. The mutations are preferably of the type that totally inactivates the protein, such as insertions, nonsense, frameshift, or deletion mutations. Any technique known in the art for producing such mutations may be used. The applicants used a transposon insertion strategy to identify the genes encoding arylsulfatase and β-glucuronidase. A deletion mutation in each gene was then constructed by homologous recombination using 5′ and 3′ DNA fragments flanking the gene which were joined together. Nonetheless, other strategies can be used to obtain the mutations in these genes. The mutations can be made directly in a gellan “production” strain, or the mutations can be transferred to such a strain from a strain in which the mutation is first made. Techniques for site-directed mutagenesis are well known in the art. See, e.g., “In Vitro Mutagenesis Protocols, second edition, Braman, Jeff, ed., Humana Press, 2002, and the commercially available QuikChangeTM kit (Stratagene). Provided with the wild-type sequences of the S. elodea arylsulfatase and β-glucuronidase genes, one of skill in the art can readily make a variety of desired mutations in these genes.
The sequence of the wild-type and mutant genes encoding arylsulfatase and β-glucuronidase have been determined. See SEQ ID NO: 1 (wild-type arylsulfatase), SEQ ID NO: 3 (deletion of arylsulfatase), SEQ ID NO: 4 (wild-type β-glucuronidase), and SEQ ID NO: 6 (deletion of β-glucuronidase). The identification of these genes and their nucleotide sequences permits one of skill in the art to readily make other mutations having the desired null phenotype for expression of these enzymes. Mutations such as insertions, deletions, nonsense, and frameshift are most likely to result in a null phenotype for the enzymes. Missense mutations can also be made and routinely tested for their effect on enzyme activity. Standard techniques of microbial genetics can be used to make suitable mutations. See, e.g., Principles of Gene Manipulation: An Introduction to Genetic Engineering, R. W. Old, S. B. Primrose, Blackwell Publishing, 1994. Standard enzyme assays can be used to test for loss of activity of the mutated enzymes. See, e.g., Kim et al., Appl Microbiol Biotechnol. 2004 February; 63(5):553-9.
Compositions of the present invention which are substantially free of arylsulfatase or β-glucuronidase contain less than 95%, 96%, 97%, 98%, or 99% of the amount of the particular protein than is contained in wild-type strains. Such a reduction in amount of enzyme should lead to sterilized milk compositions which have less than 90%, 93%, 95%, 97%, or 99% of the amount of para-cresol that is produced in compositions containing gellan from wild-type strains. The amount of arylsulfatase or β-glucuronidase protein which is produced can be measured by enzyme assay using a readily assayable substrate such as 5-bromo-4-chloro-3-indolyl sulfate (X—SO4); CAS No. 6578-07-0 from Sigma or using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GlcA); CAS No. 114162-64-0, from Sigma or RPI Corp. or using p-nitrocatechol. Alternatively the protein can be measured using an immunological technique such as a Western blot.
Gellan gum is typically used in sterilized or ultra high temperature (UHT) treated dairy products or frozen dairy products. Such products include without limitation, ice cream, frozen yogurt, pudding, whipped dairy product, coffee creamer, crème brulee, and dairy beverages. The gellan gum of the present invention can be used in these or any other foods as can typical gellan from a wild-type strain. Gellan gum is typically used for suspension of fine particles, but it also can be used to impart a favorable mouth feel.
Gellan gum can be produced using the mutant strains of the present invention according to any of the methods known in the art for wild-type strains. The bacteria are typically grown in a liquid culture medium. Such medium typically contains a carbon source, phosphate, organic and inorganic nitrogen sources, and appropriate trace elements. The fermentation is typically conducted under sterile conditions, with aeration and agitation. At the end of the fermentation period, the culture medium is collected, typically without removing the cells. The fermentation broth can be pasteurized to kill viable cells prior to recovery of the gellan gum. The gellan gum can be precipitated as is known in the art. Typically this is done with an alcohol, such as isopropanol. The precipitated gellan can be dried prior to rehydration.
Para-cresol can be measured by any means known in the art. One method which can be used employs dichloromethane extraction, concentration, and gas chromatographic-mass spectroscopy.
While the invention has been described with respect to specific examples including presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described systems and techniques that fall within the spirit and scope of the invention as set forth in the appended claims.
EXAMPLES
Example 1
The following reagents were used in screening and characterizing mutant strains:

    
    
        5-bromo-4-chloro-3-indolyl sulfate (X—SO4); CAS No. 6578-07-0 Source: Sigma.
        5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GlcA); CAS No. 114162-64-0, Source: Sigma or RPI Corp.
    
    


Example 2
The genes encoding arylsulfatase and β-glucuronidase were identified by making a library of random transposon mutants in a nonrmucoid strain of S. elodea, Gps2, using the commercially available EZ::TNTM <R6 Kgamma-ori/KAN-2> insertion kit from Epicentre (Madison, Wis.). Kanamycin resistant mutant strains were screened for lack of (or significantly reduced) blue color on selective media with specific chromogenic substrates. See Example 1. Mutants blocking arylsulfatase production or activity were identified using the chromogen 5-bromo-4-chloro-3-indolyl sulfate (X—SO4) on agar with a defined medium with chloride salts. Mutants of β-glucuronidase were selected on a defined medium with the chromogen 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GlcA).
The transposon and adjacent genomic DNA were subsequently excised from the chromosome using restriction enzymes. The restriction enzyme fragments were circularized with ligase and transformed into Escherichia coli where the transposon-containing DNA can replicate due to presence of a replicon in the transposon. Plasmid DNA was purified and sequenced. The plasmid with the gene for arylsulfatase was designated R6K-AS#14E. A portion of this plasmid has been sequenced (SEQ ID NO: 1). The plasmid with the gene for beta-glucuronidase was designated R6K-BG#6S. A portion of this plasmid has been sequenced (SEQ ID NO: 4.) These plasmids in Escherichia coli strain EC100D pir+ are being deposited at the American Type Culture Collection, Manassas, Va., on Jun. 21, 2004.
In the bacterium that had an inactivated arylsulfatase, the transposon had actually inserted in a gene for a hypothetical protein that was adjacent to the gene for arylsulfatase. In the bacterium that had an inactivated β-glucuronidase, the transposon insertion was located in the amino portion of the gene for β-glucuronidase. DNA sequencing of the genes showed that they were homologous to known genes from other species in the database of the National Center for Biotechnology Information (NCBI).
The genes for arylsulfatase and β-glucuronidase and adjacent genomic DNA were sequenced. Deletions of the genes were constructed on a plasmid and then transferred into S. elodea strains S-60 wtc and PDG-1. See WO 01/64897. The deletions were inserted in the genome by homologous recombination. DNA sequences flanking the target gene were amplified by PCR and cloned into the plasmid vector pLO2. (Lenz, O., E. Schwartz, J. Demedde, M. Eitinger and B. Friedrich. 1994, “The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation,” J. Bacteriol. 176:4385-4393.) This construct was transferred into the S. elodea strain by conjugation. The resulting kanamycin resistant strains were then grown for 30-40 generations in the absence of antibiotic. Isolates that had lost the plasmid were detected by selection for sucrose tolerance due to loss of the sacB gene on pLO2, and confirmed by kanamycin sensitivity. Isolates that had lost the plasmid after the non-selective growth were of two types, deletion or wild-type. The desired deletion strains were identified by lack of blue color on appropriate indicator agar (see example 1) and by diagnostic PCR. The scheme for construction is shown in FIG. 1 below.
To construct a precise deletion of the gene (atsA) for arylsulfatase it was necessary to determine the most likely start codon for the atsA gene and the start and stop codons of the adjacent genes. The locations of the ends of the genes were determined based on DNA sequences, homologies to other genes in GenBank and third base GC preference using the FramePlot-3 program. Ishikawa and Hotta. “FramePlot: a new implementation of the Frame analysis for predicting protein-coding regions in bacterial DNA with a high G+C content.” FEMS Microbiology Letters 174:251-253 (1999). This analysis indicated that the arylsulfatase gene is translationally coupled to the gene for the conserved hypothetical protein, i.e., the stop codon of the arylsulfatase gene overlaps with the start codon of the gene for the conserved hypothetical protein. The arylsulfatase deletion was constructed to leave the alkylsulfatase gene and the gene for the unknown protein intact, since it is not known whether these proteins are required for optimal cell growth and gellan production.
PCR primers AS5 (CCGAGCTCAACGCCTTCGACTATGTCCA; SEQ ID NO: 11) and AS6 (CCTCTAGACTGGGGATTGTCCGGAAAAG; SEQ ID NO: 12) were used to amplify a 512 bp fragment just upstream of the start codon of atsA as a SacI-XbaI fragment (total 528 bp). Primers AS3 (CGTCTAGATCCACCCCGGCGACCTTCCC; SEQ ID NO: 13) and AS4 (TATAGCATGCGGCGACCACGGGCTCCTCCTCA; SEQ ID NO: 14) were used to amplify a 479 bp fragment including the end of the atsA gene and the start of the conserved hypothetical protein as an XbaI-SphI fragment (total 497 bp). Thus, the stop codon of atsA was retained but the start codon was deleted, so no portion of the arylsulfatase protein should be synthesized. Restriction sites for cloning were added to the ends of the primers. The PCR fragments were ligated sequentially into the polylinker of plasmid vector pLO2, to form the deletion of the atsA gene. The upstream SacI-XbaI fragment was cloned into SacI-XbaI cut pLO2. Subsequently the downstream XbaI-SphI fragment was cloned (FIG. 2). This plasmid with deletion of the atsA gene was transferred by conjugation into S. elodea strains S60 wtc and PDG-1 to allow recombination of the plasmid into the chromosome. Kanamycin resistant isolates were purified, then grown in the absence of antibiotic and plated on medium with sucrose and X—SO4 to select isolates with sucrose tolerance due to loss of the plasmid-encoded sacB gene. Sucrose resistance, kanamycin sensitive, yellow colonies were selected. The atsA derivatives of S60 wtc and PDG-1 were designated GAS-1 and PAS-1 respectively.
A deletion of the gene (gusA) for β-glucuronidase was constructed on plasmid pLO2 and transferred into S60 wtc, GAS-1. The most likely start codon for the gusA gene was determined by homology to other proteins and the presence of ribosome binding sites. A region of secondary structure is upstream of the start codon. The deletion of the gusA gene was constructed to maintain secondary structures upstream and downstream of gusA. Primers Bgluc3 (AACTGCAGACACGTGGCTTGTGCCGAAC; SEQ ID NO: 7) and Bgluc4 (GGCTCTAGACTTCTCCCTGTTCCTCCGGGAAA; SEQ ID NO: 8) were used to amplify a 560 bp fragment upstream of the gusA gene as a PstI-XbaI fragment (total 577 bp). Primers Bgluc1 (TTTCTAGATGACTGTCCAGGCCCCTCTC; SEQ ID NO: 9) and Bgluc2 (TCGAGCTCCAATGTCCTCGTAGCTGTTC; SEQ ID NO: 10) were used to amplify a 489 bp fragment downstream of gusA as an XbaI-SacI fragment (total 505 bp). The PstI-XbaI fragment was cloned into PstI-XbaI cut pLO2. Subsequently the downstream XbaI-SacI fragment was cloned. This plasmid with deletion of the gusA gene was transferred by conjugation into S. elodea strains S60 wtc, GAS-1 and PAS-1 to allow recombination. Kanamycin resistant isolates were purified, grown in the absence of antibiotic and then plated on media with sucrose and X-GlcA to select isolates with sucrose tolerance due to loss of the plasmid-encoded sacB gene. A mixture of blue-green (wild-type) and light green (mutant) colonies was obtained. Sucrose resistant, light green isolates were confirmed for plasmid loss by kanamycin sensitivity. The gusA deletion derivatives of S60 wtc, GAS-1 and PAS-1 were designated GBG, GBAD and PBAD respectively.
The deletion of the gene (atsA) for arylsulfatase in strains S60 wtc and PDG-1 has been completed. The gene for β-glucuronidase has been identified and sequenced. The adjacent DNA was sequenced. A deletion of the gene for β-glucuronidase has also been constructed in each of S60, PDG-1, and GAS-1.
Samples of gellan made from strains GAS-1 (with a deletion of the gene for arylsulfatase) and GBAD-1 (with deletions of genes for both arylsulfatase and β-glucuronidase) were evaluated for p-cresol production at monthly intervals in an ultra-high temperature dairy application test. Gellan samples from the wild-type strain produced 3 to 152 (average 65) ppb p-cresol after one month and 4 to 212 (average 96) ppb p-cresol after two months. Samples of gellan from GAS-1 produced about 1 to 3 ppb p-cresol after one to five months. Samples of gellan from GBAD-1 produced less than 1 ppb (limit of detection) when tested for up to three months.

## Claims

1. A composition comprising genetically purified gellan gum substantially free of catalytically active arylsulfatase protein and substantially free of denaturing agent and substantially free of denatured arylsulfatase protein.

2. A composition comprising genetically purified gellan gum substantially free of catalytically active β-glucuronidase protein and substantially free of denaturing agent and substantially free of denatured β-glucuronidase protein.

3. A composition comprising genetically purified gellan gum substantially free of catalytically active arylsulfatase and catalytically active β-glucuronidase proteins and substantially free of denaturing agent and substantially free of denatured arylsulfatase and β-glucuronidase protein.

4. The composition of claim 1 which is a culture broth of Sphingomonas elodea.

5. The composition of claim 1 which is a precipitated culture broth of Sphingomonas elodea.

6. The composition of claim 1 which is a sterilized milk product.