Industrial Production of Xanthan Gum

Xanthan Gum Production

Industrial production of Xanthan gum started in 1970’s. Over the years it became one of the most important industrially produced anionic heterolpolyasaccharide. Reason for its prime importance is its wide use in food, cosmetics, paper making, medicine, paint and petroleum industry. It is used as a n emulsifier, thickner and friction and water mobility reducer. Xanthan gum is convenient to use over other polysaccharides due to some of its properties such as of pH resistance, salt resistance, stability towards many enzymes, viscosity properties at low concentrations and resistance to shear degradation. Xanthan gum is industrially produced by fermentation. Recombinant DNA mediated technique is used for enhancing production of xantan gum.

Areas covered:

This article covers information about industrial production of xanthan gum. This involves media, microorganisms and enzymes used for its production as well as down-stream processing of the final product.

1. INTRODUCTION

Xanthan gum is a microbial exopolysaccharide. It is mostly excreted by the bacteria of genus Xanthomonas, for example Xanthomonas Campestris NRRL B-1003, NRRL B1454, NRRL B-1043 and sphingomonas.^[1] It comprises of repeating units of pentasaccharide, which is made up of Glucose, Mannose and Glucuronic acid in the molar ratio 2:2:1.^[1,2] Its primary chain comprises of beta-d-glucose monomers and its secondary chain comprises of one glucuronic acid between two molecules of mannose.^[2] Secondary chain links primary chain at every second glucose moiety. Precursors required for production of xanthan gum includes UDP-Glucose, UDP-Glucronic acid and GDP-Mannose. Glycosyl moieties are transferred to lipid carriers with the help of glycosyltransferase. Once glycosyl moieties are transferred to the lipid carrier, acetyl and pyruvyl residues are added to it. ^[1] Length of the xanthan chain varies depending upon 1. Bacterial strain used 2. Composition of media used 3. Down-stream processing. Average Molecular weight of Xanthan Gum is 2 into 10⁶ Daltons.^[3] It can be produced by fermentation of glucose, sucrose and lactose. Various enzymes can be used for producing xanthan gum, which includes Transferase, Acetylase and Ketalase.^[2] Vectors can also be used for enhancing production both qualitatively as well as quantitatively.^[3,4] Xanthan cryogels are widely replacing Gelatin in the food industries and thus helping vegetarians. Xanthan hydrogels can be used as pH sensors.

2. AEROBIC FERMENTATION OF XANTHAN GUM USING CHEMICALLY DEFINED MEDIA:

2.1. ORGANISMS USED:

Xanthan gum can be produce by aerobic fermentation process. The organisms used for its production includes Xanthomonas Campestris primarily, along with X. Campestris other strains of xanthomonas such as X. Carotate, X. Paraveriocola, X. Translucens, X. Vasoculoeum and X. Hederae can also be used.^[4]

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The medium required for aerobic fermentation comprises of at least one carbon source from glucose, maltose and starch, water soluble nitrogen source such as peptone or yeast extract, magnesium salt, phosphate ions and other trace elements.^[5] Also a chelating agent such as citric acid is added to maintain stability of the medium in presence of iron and other trace elements. Optimum pH is 5.5-9. ^[4,5]

2.2. FERMENTATION PROCEDURE:

A seed culture is prepared by inoculating Xanthomonas in the seed culture media

Seed culture is then added to the fermentation medium (5% of the total volume)

Fermentation medium is incubated at28^oC and it is agitated (agitation speed 500 to 800rpm)

Once the cell density of more than 1gm/litre is obtained, slowly the speed of agitation is increased from 500rpm to 800rpm.

Medium is incubated further till the cell density of more than 2gms/liter is obtained. Once it is obtained, fresh media is added to the fermrntor and media from the orvious fermentation is removed.

Xanthan gum produced is precipitated by using ethyl or isopropyl alcohols.

It is further purified by cold press or filtration or centrifugation.

Presence of Xathan gum is confirmed by Mass spectroscopic analysis or NMR

Reference: [5]

The residues of the remaining media are protein rich and can be used as animal feed.^[5]

2.3 COMPOSITION OF THE FERMENTATION MEDIA:

Reference: [4,5]

3. PRODUCTION OF XANTHAN GUM USING SOLID OR SEMI SOLID MEDIA:

There may be a question in everyone’s mind. What is the need of developing a solid or a semi-solid medium for producing xanthan gum? The answer to this question is, if we use liquid media, it becomes more viscous once the production of exopolysaccharide starts. ^[6]This leads to problem in gaseous exchange. More amount solvents (Ethanol, Isopropyl alchol, KCl) are needed for precipitation of the Xanthan gum. More amount of water is required for preparing media. Energy required for aeration is more as compared to the semi-solid medium. All these factors make submerged fermentation more costly and energy utilized is more ^[6,7]. In contrast to this a semi-solid and solid media requires less amount of water, solvents required for precipitating xanthan gum are less and energy required for aeration is less. All these factors make the procedure more economical and less energy consuming.^[6,7]

3.1 USE OF POTATO OR POTATO WASTE MEDIA:

Potato or potato waste can be used to prepare a solid or a semi-solid medium. Other than potato alternative sources such as Apple pomace, spent malt grains, citrus waste, olive-mill waste and sugar beet pulp can be used for fermentation. ^[6]In this type of fermentation substrate provides complex starch medium which is utilized by the organisms. Solid medium is prepared by cutting potato or potato waste into small pieces of size ranging from ¼ inches to /3 inches.^[6] If required this medium is hydrated to certain level. However in case of a semi-solid medium, the total solid content of the medium should be 6.5% or higher. ^[6]

3.1.1 Composition of solid and semi-solid medium:

Ref: [6]

3.1.2. Organisms used:

Organisms used for this type of fermentation includes Xanthomonas and related genetically modified organism (described later in this article), which are caplable of producing Xanthan gum. Preferred strains for inoculation include Xanthomonas campestris NRRL B-1459, NRRL B-1003, NRRL B-1043. ^[6]These organisms are grown in seed culture for 3-4 days prior to inoculation in production media.^[6]

3.1.3. Fermentation procedure:

Fermentation media is produced and sterilized prior to inoculation of bacteria. Bacteria are inoculated in the medium and allowed to grow for about 72-144 hrs.^[6] this is because growth of organisms does not begin to slow down till 72 hrs and there is no significant decrease in the growth rate before 144 hrs.^[6] Medium is either incubated on a rotary shaker or inside a rolling bottle (5rpm). ^[6]Incubating it inside a rolling bottle is more energy efficient. Once the fermentation procedure is complete substrate is washed until all xanthan gum produced is dissolved in it. ^[6]This dissolved xanthan gum is then precipitated using either ethanol or isopropanol.^[6] Precipitate is then isolated either by centrifugation or filtration. Conformation of production of xanthan gum is done by Mass spectroscopy or NMR. ^[6]

3.2. USE OF DAIRY WASTE:

Dairy waste such as whey and dairy permeates can be used for producing xanthan gum. Whey refers to the liquid recovered during manufacture of cheese. And permeate is filtrate obtained when whey milk and other dairy products are filtered. ^[7] Filtration helps in removal of globular proteins such as Lactoglobulin. ^[7] Filters allow to pass lactose through it. Filtered liquid contains about 5% of solids, from which 80% is lactose. For producing xanthan gum using dairy waste, there is a need of organisms capable of utilizing lactose and producing xanthan gum.^[7] Organisms are genetically engineered either by inducing mutation or by introduction of exogenous genetic material into the genome of an organism in such a way that, it will help in converting lactose to xanthan gum.^[7] Microorganisms useful for this type of fermentation includes Xanthomonas campestris, X alblineans, X. frageriae, X. junglandis, X. mannihotis, X gummoscidans and X. aranopodis. These organisms can be genetically engineered for producing xanthan gum.^[7] X. campestris X59-1232(ATCC-55258) is known to increase the production.^[7]

4. USE OF GENETICALLY ENGINEERED ORGANISMS FOR PRODUCTION OF XANTHAN GUM.

Genetically engineered microorganisms can be used for enhanced production of xanthan gum. This will help in enhancing quality as well as quantity of an exopolysaccharide produced.^[8,9]

4.1. RECOMBINANT DNA USED FOR PRODUCING XANTHAN GUM:

Xanthomonas are obligate aerobes, which are used for production of xanthan gum. Aerobic fermentation process requires more energy for agitation.^[8] The development of recombinant plasmid helps in increasing production of xanthan gum and lowering the energy requirement.^[8] DNA sequences required for production of xanthan gum have been identified and inserted into a vector.^[8] This vector is then allowed to express in a host bacterium such as nitrifying bacterium, which do not necessarily needs aeration for its growth.^[8]

1. DNA sequences may be naturally obtained or can be created synthetically.
2. Preparation of DNA sequences as present naturally in the plasmid pRK 290-H336.
3. Insertion of DNA fragment into a cloning vector.
4. Insertion of vector into a host for cloning
5. Transfer of cloning vector into an expression host such as denitrifying bacteria.
6. Culturing of those bacteria
7. Precipitation of xanthan gum
8. Isolation of xanthan gum.

Reference: [8]

4.2. USE OF MULTICOPY RECOMBINANT DNA PLASMID FOR INCREASING PRODUCTION OF XANTHAN GUM:

A multi copy recombinant plasmid is prepared from a cloning vector pRK293 and a 12.4kb DNA fragment containing genes required for xanthan gum production. This recombinant plasmid is first inserted into a host such as E-coli for cloning.^[9] Once cloning is complete, plasmids are transferred into a xanthomonas via conjugal transfer, with the help of helper plasmid pRK2013 (ATCC-37159). This plasmid is a high copy number plasmid, which increase the production of xanthan gum. ^[9]

5. ENZYMES USED IN PRODUCTION OF XANTHAN GUM:

Enzymes used in producing xanthan gum include Acetylase, Ketalase, Pyrophosphatase and Transferase. The functions of these enzymes are mentioned in the figure below

It has been observed that non-acetylated and non-pyruvylated polymers are more viscous than acetylated and pyruvylated polymers, even when used in smaller quantities.^[10] Certain strains of Xanthomonas are genetically engineered to produce non-acetylated and non-pyruvylated polymers. This helps in improving quality of final product.^[10]

6. USE OF PROTEASES AND LYSOZYMES IN RECOVERY OF XANTHAN GUM.

WHILE isolating xanthan gum from fermented broth it is first dissolved completely and then precipitated using alcohols.^[11] The problem in this process is that, a clear solution is not usually obtained while dissolving. Therefore for obtaining clear solution after dissolution, Proteases and Lysozymes may be used.^[11] This helps in complete recovery of xanthan gum without any loss. The broth is preheated prior to protease treatment. The broth is treated with Lysozyme after treatment with protease. Once the clear solution is obtained Xanthan gum may be precipitated using ehanol or isopropanol.^[11]

7. SPHINGOMONAS USED IN PRODUCTION OF XANTHAN GUM.

Xanthan gum can be produced by the strains of Sphingomonas . 12 genes required for synthesizing xanthan gum. These genes include gumB,C,D,E,F,G,H,I,J,K,L,M.^[12] all these genes are clustered on one DNA segment and then inserted in a expression vector. This vector is then allowed to express in a strain of Sphingomonas.^[12] Xanthan gum produced by Sphingomonas is indistinguishable from that of Xanthomonas.^[12]

8. CONCLUSION:

Xanthan gum is the one of the most important industrially produced microbial exopolysaccharide. Different strategies have been used for enhancing production as well as the the down-stream processing of xanthan gum.^[1] Use of solid and semi-solid media is more economical and energy efficient than the liquid media, as the energy required for aeration is less and less alcohol is required for precipitation.^[4] Potato waste and dairy permeates can be used for producing xanthan gum. Xanthan gum obtained from dairy permeates is of better quality than the xanthan obtained from potato waste.^[6,7] This is because absence of globular proteins in the production media. Recombinant DNA technology can be used for producing xanthan gum.^[8] We can insert a recombinant vector expressing genes for xanthan gum production into a nitrifying bacterium. This helps in increasing production as well as lowering the cost for production.^[9] Multicopy recombinant plasmids can be used for increasing production of xanthan gum.^[9] These plasmids are first allowed to express in host such as E-coli then they are transferred into xanthomonas by conjugation. Enzymes can be used for enhancing the production.^[9] These enzymes include Acetylase, Ketalase, Acetyltransferase.^[10] Xanthan gum can be produced by using strains of Sphingomonas. 12 genes required for producing xanthan gum can be cloned into Sphingomonas. These genes include gumB,C,D,E,F,G,H,I,J,K,L,M.^[12]