INTRODUCTION:

Pullulan is a fungal exopolysaccharide produced by Aureobasidium pullulans. It consists of linear arrangements of maltotriose units. It has alpha (1->4) and alpha (1->6) glucan (polysaccharide of D-glucose) monomers linked by glycosidic bonds. The maltotriose units are linked by alpha (1->4) glycosidic bonds; whereas consecutive units are linked to one another by an alpha (1->6) glycosidic bond. The pigmented pullulan exopolysaccharide is decolourized by using activated charcoal (Some amount of pullulan is lost by it)^[1] It can be represented as (C₆H₁₀O₅)_x.^[2]

Structurally similar to amylopectin in starch and maltodextrin except the relative ratio of alpha (1->4), alpha (1->6) bonds, the tertiary structures of the molecule, the extent and mechanism of degradation of the materials in the human gut. It can be classified in the group of soluble fibres^[3]

It is hydrophilic in nature because of the presence of approximately 9 -OH groups per repeating unit of maltotriose.

It is water soluble and is insoluble in inorganic solvents except dimethylformamide and dimethyl sulphoxide.^[4] It is produced aerobically. It is not hygroscopic, has adhesive properties, compression moldings, and strong, has the ability to form fibres and oxygen-impenetrable films. It is non-poisonous, non-immunogenic, non-mutagenic and non-cancerous in nature.^[5] Pullulan is superior to starch in water retention and hence spoilage by drying is lesser in comparison.^[6] It is stable over a wide range of pH conditions in the solution. It doesn’t release any harmful gas even when burned and is spontaneously broken down by microbes even when scrapped as it is. The exopolysaccharide, pullulan, also resists the penetration of oxygen and this feature is being exploited in the food-packaging industry. It is moldable and spinnable non-toxic, edible, and biodegradable.^[7]

An undesirable distinctive trait of A. pullulans is the production of a dark pigment, which is a compound resembling melanin and seems dark greenish to black in colour.^[8] A. pullulans is imperfect genetically and conventionally has been considered to be among the group of imperfect fungi or Deuteromycetes.^[9]. When made hydrophobic by acetylation, polymers will self-associate to form nanoparticles that will encapsulate hydrophobic drugs.^[10]

MATERIALS AND METHODS:

Collection of sample:

Brahmapuram (see Fig.1), a small hillock in Vellore has the optimum conditions for the growth of pullulan producing fungus. Leaves of different plants were collected in airtight ziplock bags at three different altitudes-low (at the foothills), medium (from creepers) and at the top. Collected leaves and flowers were put in 50 ml of sterile water for two days. Samples from different regions were put in different sterilized conical flask, in aseptic conditions.

Selective enrichment procedure:

The media was prepared with the following components:

Table 1: Components of the media

Salt	Amount
(NH₄)₂HPO₄	0.5g
NaCl	0.025g
MgSO₄.7H₂O	0.025g
K₂HPO₄	1g
Sucrose	5g
FeSO₄	0.05g
MnSO₄	0.05g
ZnSO₄	0.05g
Distilled water	500ml

The media was adjusted to pH 4 by adding 1M K₂HPO₄ and 1M KH₂PO₄ and was then sterilized. Antibiotics, streptomycin and chloramphenicol, 20 mg/ml each, were added to inhibit the growth of undesired yeast like fungi.

The supernatant from the sterile water, in which the leaves were kept for two days, was added to the media prepared earlier, and kept in the mechanical shaker for three days at 100 rpm.

After 3 days, the culture was plated. The aforementioned media was used without the antibiotics. 2% agar was added in addition to all the ingredients already mentioned. The petri plates were incubated at 25 degrees Celsius for 3 days.

Spread Plate Technique:

After three days, the fungus could be seen along with its product. This product was extracted. Spread plate technique was used to get maximum product from a high concentration of yeast like fungus.

Leghaemoglobin:

Leghaemoglobin has a high affinity towards oxygen, near about 10 times higher than the Beta chain of haemoglobin of humans. This ensures low oxygen concentration for the oxidation of the product. In Rhizobium, the leghaemoglobin ensures enough oxygen concentration for the respiration of the bacterium. Leghaemoglobin is an oxygen scavenger. It was added to prevent repeated oxidation of the product obtained.

Collection and incorporation of leghaemoglobin:

Groundnut plants were collected from a nearby farm. Then the nodules were picked from the roots. The nodules so obtained were washed with water to remove soil particles. The nodules were crushed with the help of a mortar and a pestle. The paste formed was mixed in a buffer solution (which was prepared separately using acetic acid and sodium acetate) of pH 4. 164 ml of 0.1M Acetic acid and 36 ml of 0.1M Sodium acetate was used to prepare the buffer solution of pH 4. The entire solution was centrifuged at 5000 rpm for 20 minutes (see Fig. 2), to separate the soil particles from the leghaemoglobin in the buffer. The supernatant (which contains the leghaemoglobin) obtained was used henceforth.

The extracted product was taken in a sterilized petri plate in sterile conditions and leghaemoglobin was added to the petri plate to avoid oxidation of the product. Then we put it in the oven at 60 degrees celsius for 2 days.

Spectrophotometric Analysis:

The optical density of the culture was measured at regular intervals after a couple of days, after the addition of leghaemoglobin to the culture media.

Antibiotic Sensitivity Test (ABST):

Antibiotic Sensitivity Test (ABST) was conducted for the yeast like fungus. Agar media was prepared using Mueller Hinton agar and agar-agar in a 250 ml conical flask. Mueller Hinton agar is used for ABST because of its characteristic properties that allow easy penetration of the antibiotics into the media. The prepared media and two petri plates were sterilised. The media was added to the two petri plates in a Laminar Airflow chamber and then allowed to settle. One of the petri plates was used as a control and culture was added to the other petri plate. A cotton swab was dipped into the culture and spread evenly on the agar surface. Five selected antibiotic discs along with a control disc (paper) were then added on both the plates and were carefully spaced from each other. The antibiotics that were selected include:-

· Azithromycin - 30 mcg (AT 30)

· Gentamycin - 10 mcg (GEN 10)

· Pristinamycin - 15mcg (RP 15)

· Cefotaxime - 15 mcg (CTX 15)

· Co-trimoxazole - 25 mcg (COT 2a5)

The plates were then sealed using paraffin tape and kept in an incubator at 37^oC for two days. After two days the plates were observed for zones of inhibition which were measured in order to analyse the effectiveness of the antibiotics on the yeast like fungus. (The setup is shown in Fig 7)

Lactophenol cotton blue staining test:

The lactophenol cotton blue staining test was performed for confirming that the yeast like fungus producing the pullulan is a fungus. The slide was observed under a microscope. (refer to Fig 4)

GC-MS Analysis:

GC-MS analysis is a combination of gas chromatography and mass spectroscopy. The sample is first vapourized and made to pass through a capillary column with the ‘stationary phase’ coated on the interior of it. This finds out the retention time which in turn is helpful in the identification of the compound. Retention time for every compound is different depending on its interaction with the stationary phase. The sample solution is introduced to the Gas Chromatography inlet where the sample solution vapourizes and sweeps onto a chromatographic column by the helium gas, which is typically the carrier gas. The sample sweeps through the column and the compounds consisting of the desired mixture is then segregated in accordance with their relative interaction with the column coating and the Helium gas, which is typically the carrier gas. The terminal portion of the column is then made to go through the heated transfer line that terminates at the passageway to the source of ions, where the compounds moving out from the column are ionized.

RESULT AND DISCUSSION:

Lactophenol cotton blue staining test:

Upon observing the stained slide under the microscope, we were able to notice many of the fungal characteristics such as hyphae and sporangia (see Fig. 4).

Two varieties of pullulan were found, from the duplicates of the culture obtained from the top of the hill. One appeared greenish, due to the presence of melanin produced by the strain of fungus which had a high yield of pullulan as well as melanin (see Fig. 5). The other appeared yellowish-white produced by a strain of fungus which had a high yield of pullulan but low yield of melanin (see Fig. 6).

The product that was extracted appeared yellowish-white in colour and nearly spherical in shape. The pigmented product was a cumulation of several small, green, spherical ball like structures.

Spectrophotometric Analysis:

The O.D. values were constant and showed no increase whatsoever. This shows that the desired fungus did not grow in the anaerobic conditions established by the use of leghaemoglobin. Thus, this proves that the fungus is an obligate aerobe.

Antibiotic Sensitivity Test:

The ABST that was performed yielded the following results: -

Table 2: Zone of inhibition of different antibiotics on pullulan

Name of antibiotic	Diameter of zone of inhibition (in cm) (Approximately)
Azithromycin - 30 mcg (AT 30)	3
Gentamycin - 10 mcg (GEN 10)	2.7
Pristinamycin - 15mcg (RP 15)	2.9
Cefotaxime - 15 mcg (CTX 15)	3
Co-trimoxazole - 25 mcg (COT 25)	4.1

Gas Chromatography-Mass Spectroscopic Analysis:

Gas Chromatography Mass Spectroscopic (GC-MS) Analysis had been carried out for the product obtained. The following picture shows the result.

Graph 1: Shows low melanin pullulan (first peak)