Polysaccharides

Biopolymers (polyhydroxyalkanoates)

POLYSACCHARIDES

Microbial polysaccharides are used for food, pharmaceutical, and medical applications: this wide range of usefulness derives from the great diversity in structural and functional properties.

Applications of commercially available microbial polysaccharides including xanthan, xylinan, gellan, curdlan, pullulan, dextran, scleroglucan, schizophyllan, and cyanobacterial polysaccharides

The commercial value of polysaccharides is based on its ability to modify the flow characteristics of solutions (Rheology). They can incr viscosity and hence used as thickening and gelling agents.

• Polysaccharides made by microrganisms are secreted from the cell to form a layer over the surface of the organism, often of substantial depth in comparison with the cell dimensions (Figure 1).

Because of their position they are characterized as exopolysaccharides, to distinguish them from any polysaccharides that might be found within the cell.

The functions are thought to be mainly protective, either as a general physical barrier preventing access of harmful substances, or more specific as a way of binding and neutralizing bacteriophage. In appropriate environments they may prevent dehydration.

Exocellular or ‘capsular’ polysaccharide layer (labeled P) from Streptococcus pneumoniae

• Prevent phagocytosis by other micro- organisms or the cells of the immune system. The capsular polysaccharides (CPSs) are often highly immunogenic, and may have evolved their unusual diversity as a way of avoiding antibody responses: advantage of this feature can be taken in the development of vaccines.

• adhesion and penetration of the host; Plant lectins (glycoproteins) that have specific binding properties with respect to carbohydrate structures; general defense of plants against bacterial infection.

• Polysaccharides can be involved in pathogenicity. Pseudomonas aeruginosa, commonly found in respiratory tract infections, produces alginate which contributes to blockage in the respiratory tract

• polysaccharide biotechnology: xanthan, priced at about US$14 per kg, used mainly in food applications, to cyclic dextrans, valued at about US$50perkg and used in high-value applications in research and pharmaceuticals

• Certain microbes are known to produce nearly all the major plant polysaccharides such as glucans, alginate-like materials, and even cellulose – as well as the complex bacteria-specific materials.

Production

Batch cultureExcess of carbon supplyCarbon/nitrogen ratio of 10:1When acidic PS limited polysaccharides are synthesized90% oxygen saturation

Biosynthesis

Starting with glucose or appropriate sugar and several enzymatic reactions,

Recovery

Marked increase in viscosity of culture brothCan be precipitated by salts, acids or organic solvents

Microbial PSPlant PS

CheapUncontrolledOccurs for a short duration

20 PS of commercial adv and valueDependent of rheological property

Only microbial polysaccharide currently produced on a Large scale: XANTHAN

XANTHAN: polysaccharide polymer produced by Xanthomonas campestris

Long chain polysaccharide composed of the sugars glucose, mannose, and glucuronic acid. The backbone is similar to cellulose, with added sidechains of trisaccharides (3 sugars in a chain)Gram negative bacteria, plant pathogen causes black rot in Brassica (cauliflower and broccoli) produces a slimy gel that protects bacteria from viruses and prevents it from drying out.

Has very high viscosity and is stable at extreme physical and chemical environments, shows physical and chemical properties like plastic, used in many ways: eg. oil drilling

Production: 20,000 tonnes per year

Pentasaccharide: Glu-man-glcA-Ac-PyrBranched polymer with b-1,4 linked glucan (glucose polymer) backbone bound to trisaccharide (Man, GlcA, Man). Man has Acetate or Pyruvate groups (number is variable dependent on bacterial strain)Viscosity is denpendent on contents of pyruvate and acetate

E415

B-glc (1-4)-B-Glc (1-4)

1-3

b-Man-(1,4)-b-GlcA- (1-2)-a-Man-6-O-AcTrisaccharide chain

Glucan backbone

Pyr4 6

Commercial production of Xanthan Gum

X. Campestris should be grown on a cheap and abundant source of carbonWild type can use glucose, sucrose and starch but not lactose

Whey is a waste product of cheese making industry used as filler in several foods 95% water, 3.5-4% lactose and small amounts of proteins, minerals LMW organic cpds

Due to lactose intolerance alternatives are requiredDisposal of whey in rivers can lead to depletion of dissolved O2Transporation of whey to landfill sites is exceptionally expensiveGroundwater leaching and contamination a concern

Only advantage is that it can be used as a carbon source for growing industrial MO

Genetic engineering of X. campestris to grow on WHEY

E.coli lacZY (b-galactosidase and lac permease)cloned to broad host range plasmid under transcriptional control of X. campestris bacteriphase promoter. Introduced in E.coli and transformed to X. campestris by tripartite mating

Transformants wgich maintained the plasmid expresses lacZY and used lactose as sole carbon, produced hih levels of xanthan gum with glucose, lactose or whey as carbon source.

Compared to wild type which produced xanthan only when grown on glucose

Helped to convert a nuisance waste product into a substrate for production of economically valuable bopolymer.

DEXTRANS They are glucans (polymers of glucose)

A(1-6) linkages some have a-1,2 and a1,3, and 1,4MW is 15,000-500,000

Applications: Blood plasma expanders for prevention of thrombosisWound healing and dressingpurification of biomolecules

Production Microorganisms like Leuconostoc mesentroidesThey are produced by extracellular enzymes like dextransucrase which act on sucrose and bring polymerization of glucose residues and liberates free fructose in medium

Batch fermentation

Multi-celled Algae (divided into several groups across classification)␣ Have a similar function as plants do on land␣ Photosynthesize, base of food chain, provide habitat for other organisms␣ Most live attached to rocks ␣ Structures are different from plantsDivision (Phylum) Chlorophyta Green algaeDivision (Phylum) Rhodophyta Red AlgaeDivision (Phylum) Phaeophyta Brown algae

Algae Structures

Photosynthesis, takes up water and nutrients

Buoyancy: air filled, allows algae to float in water

Photosynthesis, takes up water and nutrientsAttaches algae to rock, doesn’t grow into rock, only for attachment

Seaweed and marine algae have been valued for centuries in Asia and the Pacific Islands for their nutritional and healing properties as they are packed with potassium, vitamins A, B, C, D, and E.

They are very high in iodine content and are used to treat some thyroid conditions.

A brown seaweed (kelp), is found to have antibacterial and antiviral properties and has been extensively used in clinical trials to lower blood pressure in heart patients.

Several marine seaweeds and submerged vegetation in seawater are reported to have antagonistic activity and have been found effective against various viruses.

Polysaccacharides from marine environment

Purple layer contains a sulphated polysaccharide called Porphyran, which is a complex galactan, and has shown higher gelling capacity reported to inhibit the growth of Sarcoma 180 tumors in mice.A substance named porphyosin isolated from Porphyra exhibited anti ulcer activity.

red alga Porphyra umbilicalis

4-linked 6-O-sulfo-α-L-galactopyranose residue(the biological precursor of the 3,6-anhydro residue (agarose)

3-linked 6-O-methyl-β-D-galactopyranose residue

Agar polysaccharides are effective against poliovirus, herpes simplex, dengue viruses, etc.

Agar: well known gelling agent. Gelidium spp. Gracilaria (Red seaweeds (Rhodophyceae)Agarose

Agaropectin

(1 4)-3,6-anhydro-α-L-galactopyranosyl-(1 3)-β-D-galactopyranan

Agarose is purified form of agar. Both have variety of uses in biochemistry, molecular biology, microbiology.

Used as gelling agent for meats, confectionery, icing stabilizer in baked goods in food industry

Used as laxative (as it cannot be metabolized) in medicinal applicationsAlso as flexible moulds in dentistry and criminology

RED ALGAE

BROWN ALGAE

Alginates soluble in water and form gels with addition of calcium or bivalent ions

Phaeophyceae

Laminiaria spp. Sargassam spp.

Calcium ions form a link b/w G moleculesIf ions removed gel will break

Alginate gels cannot be reversed and are not stable at low pH. But high viscosity and hydration make them useful for salad dressings, frozen foods, icings and film formation.

Textile and paper industry uses to thicken inks, coat papers and reduce staining

Enzyme immobilization

Calcium alginates provide a moist healing environment by converting the exudate into a gel. A reaction between the calcium in the dressing and the sodium in the wound exudate results in a chemical ion exchange, which forms a gel-like substance. The gel conforms to the wound, providing a soft, moist healing environment. Due to it’s dehydrating effect, this dressing should not be used with dry wounds.

Also shows hemostatic activity

30000 tonnes per year and supply much more than this

Sorbsan is made from seaweed into delicate fibers and cut into unwoven sheets

Sorbsan can be easily separated and fluffed up before placing it into a

wound bed

RED ALGAERhodophyceaeCarrageenans First in Ireland

Carrageenans are ideal food additives: they have a range of gelling and emulsifying properties ranging from a soft slime to a brittle gel that one could nearly walk upon.

They also have a high reactivity with a range of materials including, most importantly, milk proteins, being widely used at low concentrations in dairy products to prevent fractionation of milk constituents. 

In fact, a major application is found in chocolate milk, a very popular daily drink in the USA and Europe, and now spreading elsewhere.

Carrageenans are also very good at keeping chocolate in suspension.

Best quality from Philippines

SCLEROGLUCAN GLUCOSE POLYMER (glucomer)

Neutral polysaccharide with b-1,3 glucan backbone and single glucose residue branches b (1,6)Branching occurs at a regular sequence at every 3rd glucose unit in the polymer backbone chain

Scleroglucan is a fungal hexopolysaccharideUseful for stabilizing latex paints, printing inks and drilling muds

GELLAN Linear heteopolysaccharide2glu, 1GlcA and one rhamnosePseudomonas elodea

A deacetylated gellan forms fim brittle gels : GELRITE (US company)

Used in food industry as low temperature thickner

POLLULAN

CURDLAN

A GLUCOSE POLYMER (a-glucan) with a-1,4 and few a 1,670% of glucose is converted to pollulan

A GLUCOSE POLYMER (b-glucan) glucose hed by b-1,3

Alcaligenes faecalis, Rhizoium trifolli, Agarobacterium rhizogenes

Fungus: Aurobasidium pollulans

Forms strong gels ehen heated to 55oC, gelling agent, immuno modulator and immobilization of enzymes

Pectin

Pectin is a long chain of pectic acid and pectinic acid molecules. Because these acids are sugars, pectin is a polysaccharide. It is prepared from citrus peels and the remains of apples (pomace) after they are squeezed for juice.

In the plant, pectin is the material that joins the plant cells together. When fungus enzymes break down the pectin in fruit, the fruit gets soft and mushy.

During ripening, pectin is broken down by the enzymes pectinase and pectinesterase; in this process the fruit becomes softer as the cell walls break down.

α-(1-4)-linked D-galacturonic acid

•Pectin is a thickener in many products. If there is sufficient sugar in the mixture, pectin forms a firm gel.

•Jams and jellies are thickened with pectin. Pectin binds water, and thus keeps products from drying out. It stabilizes emulsions.

•Pectin combines with the calcium and whey proteins of milk, stabilizing foams and gels made with cream or milk.

•In the cigar industry, pectin is considered an excellent substitute for vegetable glue and many cigar smokers and collectors will use pectin for repairing damaged tobacco wrapper leaves on their cigars.

•Pectin is also used in throat lozenges as a demulcent. In cosmetic products, pectin acts as stabilizer. Pectin is also used in wound healing preparations and specialty medical adhesives, such as colostomy devices

•Pectin is not digested, and is considered a beneficial dietary fiber. In human digestion, pectin passes through the small intestine more or less intact. In the large intestine and colon, microorganisms degrade pectin and liberate short-chain fatty acids that have positive influence on health (prebiotic effect)

Uses

Ghum or Gum MaterialsNatural gums are polysaccharides of natural origin, capable of causing a large viscosity increase in solution, even at small concentrations. In the food industry they are used as thickening agents, gelling agents, emulsifiers and stabilisers.

E numbers are number codes for food additives and are usually found on food labels throughout the European Union1. Agar (E406), obtained from seaweed

2. Alginic acid (E400), from seaweed 3. Beta-glucan, from oat or barley bran 4. Carrageenan (E407), from seaweed 5. Chicle gum, an older base for chewing gum

obtained from the chicle tree 6. Dammar gum, from the sap of

Dipterocarpaceae trees 7. Gellan gum (E418), produced by bacterial

fermentation 8. Glucomannan (E425), from the konjac plant 9. Guar gum (E412), from guar beans 10. Gum arabic (E414), from the sap of Acacia

trees 11. Gum ghatti, from the sap of Anogeissus

trees 12. Gum tragacanth (E413), from the sap of

Astragalus shrubs

13. Karaya gum (E416), from the sap of Sterculia trees

14. Locust bean gum (E410), from the seeds of the carob tree

15. Mastic gum, a chewing gum from ancient Greece obtained from the mastic tree

16. Psyllium seed husks, from the Plantago plant 17. Sodium alginate (E401), from seaweed 18. Spruce gum, a chewing gum of American

Indians obtained from spruce trees 19. Tara gum (E417), from the seeds of the tara

tree 20. Xanthan gum (E415), produced by bacterial

fermentation

Beta-glucan, from oat or barley bran

polysaccharides occurring in the bran of cereal grains, the cell wall of baker's yeast, certain types of fungi, and many kinds of mushrooms.

The cereal based beta-glucans occur most abundantly in barley and oats and to a much lesser degree in rye and wheat.

They are useful in human nutrition as texturing agents and as soluble fiber supplements, but problematic in brewing as excessive levels make the wort too viscous.

Yeast derived beta glucans are notable for their immunomodulatory function. The differences between soluble and insoluble beta glucans are significant in regards to application, mode of action, and overall biological activity.

Beta 1,3-D glucans are being referred to as biological response modifiers because of their ability to activate the immune system.

However, it should be noted that the activity of Beta 1, 3-D glucan is different from agents that stimulate the immune system.

Agents that stimulate the immune system can push the system to over-stimulation, and hence are contraindicated in individuals with autoimmune diseases, allergies, or yeast infections.

Beta 1, 3-D glucans seem to make the immune system work better without becoming overactive. They accomplish this by activating phagocytes, which are immune system cells whose function is to trap and destroy foreign substances in our bodies such as bacteria, viruses, fungi, and parasites.

In addition to enhancing the activity of phagocytes, beta-1,3 glucans also reportedly lower elevated levels of LDL cholesterol, aid in wound healing, help prevent infections, enhance NK cell function, and help in the prevention and treatment of cancer

Gellan gum: is a water-soluble polysaccharide produced by Sphingomonas elodea

Also known commercially as Phytagel™ or Gelrite®, is used primarily as a gelling agent, alternative to agar, in microbiological culture. It is able to withstand 120 °C heat, making it especially useful in culturing thermophilic organisms. One needs only approximately half the amount of gellan gum as agar to reach an equivalent gel strength, though the exact texture and quality depends on the concentration of divalent cations present

Chicle gum is the natural gum from Manilkara chicle,Wrigley Company was a prominent user of this material, today there are only a few companies that still make chewing gum from natural chicle. This is because by the 1960s chicle was replaced by butadiene-based synthetic rubber which was cheaper to manufacture.

Dammar Gum or Jhuna used as room freshner, control of mosquitoes and in worshiping

Glucomannan is mainly a straight-chain polymer, with a small amount of branching.

The component sugars are β-(1→4)-linked D-mannose and D-glucose in a ratio of 1.6:1.

The degree of branching is about 8% through β-(1→6)-glucosyl linkages.

Konjac

Guar gum, also called guaran, is a galactomannan. It is primarily the ground endosperm of guar beans.

The guar seeds are dehusked, milled and screened to obtain the guar gum.

It is typically produced as a free flowing, pale, off-white colored, coarse to fine ground powder.

Guar gum retards ice crystal growth non-specifically by slowing mass transfer across the solid/liquid interface. It shows good stability during freeze-thaw cycles

Psyllium seed husks,

plantagoIsabgol

Animal Biomass: Chitin

Chitin is a long-chain polymer of a N-acetylglucosamine, a derivative of glucose. It is the main component of the cell walls of fungi, the exoskeletons of arthropods, such as crustaceans (like the crab, lobster and shrimp) and the insects, including ants, beetles and butterflies, the radula of mollusks and the beaks of the cephalopods, including squid and octopuses.

Chitin has also proven useful for several medical and industrial purposes. Chitin is a biological substance which may be compared to the polysaccharide cellulose and to the protein keratin. Although keratin is a protein, and not a carbohydrate like chitin, both keratin and chitin have similar structural functions.

N-acetylglucosamine

b-1,4 linkage

Chitin is the second most abundant polysaccharide in nature (after cellulose). At least 10 gigatons of chitin are synthesized and degraded each year in the biosphere.

Chitin is translucent, pliable, resilient and quite tough. In arthropods, however, it is often modified, becoming embedded in a hardened proteinaceous matrix, which forms much of the exoskeleton.

In its pure form it is leathery, but when encrusted in calcium carbonate it becomes much harder

Chitin is one of many naturally occurring polymers.

Its breakdown may be catalyzed by enzymes called chitinases, secreted by microorganisms such as bacteria and fungi, and produced by some plants.

Some of these microorganisms have receptors to simple sugars from the decomposition of chitin.

If chitin is detected, they then produce enzymes to digest it by cleaving the glycosidic bonds in order to convert it to simple sugars and ammonia.

Chitosan (a more water-soluble derivative of chitin). It is also closely related to cellulose in that it is a long unbranched chain of glucose derivatives.

Commercially derived from shrimps

Preparation of chitin and chitosan

Biotechnological Uses of Chitinolytic enzymes

Chitin has a broad range of applications in biochemical, food, and various chemical industries.

It has antimicrobial, anticholesterol and antitumor activities

Chitin and its related materials are also used in wastewater treatment drug, wound healing, and dietary fiber

Industrial (water purification, stabilizer, pharmaceuticals)Medicinal (surgical threads, wound healing, role in immune response

to allergic diseases)Agricultural (inducer of plant immune system and hence enhance

defense mech.; fertilizer

In vertebrates, chitinases are usually part of the digestive tract.

In insects and crustaceans, chitinases are associated with the need for partial degradation of old cuticle.

Implicated in plant resistance against fungal pathogens because of their inducible nature and antifungal activities invitro (Chitinase in fungi is thought to have autolytic, nutritional, and morphogenetic roles. In viruses, chitinases are involved in pathogenesis

In bacteria, chitinases play a role in nutrition and parasitism.

In addition to the above potential applications,chitinases can be used for the production of chitooligosaccharides, which have been found to function as antibacterial agents, elicitors of lysozyme inducers, and immunoenhancers

Chitinases can also be used in agriculture to control plant pathogens

Chitinases