Fermentation Assisted Agarwood Oil Production Method

Field of Invention

The present invention relates to fermentation assisted fragrant agarwood oil production. More particularly the present invention relates to the fragrant sesquiterpenoid agarospirol (a major component of Khara Oil) production using fermentation. Even more particularly the present invention utilizes spores of Fusarium for fungus assisted fermentation of agarwood for the production of Agarospirol (component of Khara Oil).

Background of Invention:

Agarwood has high demand throughout the world as a raw material for incense, perfume and medicine purposes, with Middle East and East Asia as the two major regions of consumption (Antonopoulou et al., 2010). As the wealth of the consumer countries has gradually increased in the recent decades, the market’s demand for agarwood started to exceed its supply. Global agarwood prices can be ranging from US$ 20 - 6,000 per kilogram for the wood chips depending on its quality or US$ 10,000 per kilogram for the wood itself (Abdin, 2014). In addition, the value of agarwood essential oil can be as high as US$ 30,000 per kilogram. The annual global market for agarwood has been estimated to be in the range of US$ 6 - 8 billion (Akter et al., 2013), yet a large number of the trades have not been recorded.

Wai Wai Than (2013) examined the fungal diversity present in the agarwood samples of Myanmar. The oleoresin like in any other places is extensively valued as a therapeutic and aromatic compound in Myanmar. Fungi from different agarwood samples were isolated and characterized. The resin content was characterized and related to the presence of fungi in each. It was found that the presence of different fungi on wood influenced the quality and content of oleoresin in wood. Different strains like Aspergilus, Fusarium, Alternaria and Nectria were some of the species they found associated to the formation of agarwood.

One obvious advantage of using fungal inoculum is that it is generally believed to be safe for handling and eco-friendly. However, fungal inoculation will normally give rise to localized and inconsistent quality of agarwood due to the different fungal consortium used. As a solution, laborious holing process and long incubation time is required to maximize the colonized surface area on the tree to produce better quality of agarwood (Mohamed et al, 2014).

There are certain drawbacks in the above-mentioned prior arts mainly, the prior arts are talking about growing the fungus on the tree in itself for which a fungal consortium is required to spread over the surface area of plant and that is one of the limitations i.e. even spread of inoculum so that better quality of agarwood is produced and it is not addressing the actual production of essential Khara oil. (Agarospirol).

The prior arts on agarwood- fungus interaction only talk about the role of fungus in inducing resin development in the field where the tree is infected by the fungus. However, after harvesting and chipping of agarwood resinous parts, a soaking step akin to fermentation is practiced. The microbial scenario featuring fungus assisted fermentation can be interesting and remunerative.

Protocols for the fermentation of agarwood chips to obtain the desired quality of agarwood oil are available. However, most of the approaches talk about fungi on wood.

The present traditional process does not factor in the fermentation/soaking as a step that can impact the quality and quantity of agarwood oil produced. The traditional process has a wide time range of fermentation (45-90 days) and varies widely with type of resinous wood. Due to prolonged soaking, the raw material remains in the soaking for a long duration thereby limiting the number of distillations possible per year. This limits the farm income. Also, the traditional soaking since it is not backed by scientific R&D. which leads to suboptimal yields and impacts quality. Other processing methods (pulverization, enzyme treatment, etc.) have not been successful yet in agarwood oil processing.

Fungus assisted fermentation is useful since it mimics the natural fermentation/soaking process and is a fully organic solution without any genetically modified organism or chemical additives in the process. Fungi are known to be associated with agarwood formation upstream (infection and resin development). Their potential in the downstream particularly in quality and quantity enhancement of oil is a strong possibility. Fungi are endowed with enzymes that act on wood tissue naturally. Therefore, fungus assisted fermentation is the choice for the production of agarwood oil.

Fungus assisted fermentation saves time compared to traditional process by its enzymatic action and increases productivity thereby saving raw material (agarwood chips) and energy (firewood, rice husk). Fungus assisted fermentation is scalable therefore can be incorporated into existing process and scale of traditional oil production.

Agarwood oil production has stagnated for a long time due to lack of R&D. The downstream of agarwood production bioprocess has remained neglected from the point of view of R&D as mostly focus is on upstream agarwood formation/production.

Hence there is a need to provide a method by which any quality of agarwood can be treated effectively to extract the active ingredient i.e. Agarospirol in case of agarwood oil in lesser amount of time and which is cost effective. The above-mentioned drawbacks can be overcome with the fungus assisted fermentation of agarwood.

Objects of the Invention

The primary objective of the present invention is to produce agarwood oil with the help of fungus assisted fermentation.

Another object of the present invention is to provide a method of fungus assisted fermentation for production of agarwood oil.

Yet another object of the present invention is to provide a method of fungus assisted fermentation for production of agarwood oil which is optimized to suit the fungal strain.

Still another object of the present invention is to provide a method of production of optimized quality of agarwood oil.

Summary of the Invention:

Accordingly, the present invention provides a fungus assisted fermentation method for agarwood oil production. The proposed output of the fermentation assisted fragrant agarwood oil production is to target the critical soaking step in the traditional production of agarwood oil. The prolonged soaking/fermentation of wood (>45 days) in water prior to distillation has a profound effect on the quality of oil. Although it is recognized by agarwood producers it has neither been factored in production nor studied scientifically. Replacing the conventional method by fungal fermentation improved recovery and quality parameters of oil and by yielding more agarospirol in lesser duration of time. Since agarospirol was used as a reference for quality. After 14 days of fermentation a significant increase in agarospirol levels across fungal fermentation conditions were observed. This indicated a qualitative improvement induced by fermentation. Further an actual comparison of the agarospirol levels with reference Khara oil showed that fermentation for 14 days yielded agarwood oil with 29.7% of agarospirol closer to Khara oil (35%) which is commercial standard and which is a more concentrated oil compared to the experimental yield.

Detailed description of the invention:

Accordingly, the present invention provides a method for enhanced quantity and quality of the agarwood oil in lesser time with the help of fermentation as follows:

1. Sanitizing the agarwood chips containing fragrant resin by sterilization at 80°C in a semi- closed autoclave and followed by UV exposure in a laminar hood chamber for 20 mins.

2. A Fusarium is used for fermentation.

3. Adding steam sterilized 270 gm of resinous agarwood chips in 180 ml of sterile water characterised to be moisten the wood

4. Adding autoclaved 270 gm of resinous agarwood to 180 ml of fungal 10 suspension preferably of concentration 1x106 spores/ml in sterile water in a separate setup;

5. Mixing uniformly and fermenting in fermenter device for 14 to 35 days

6. Maintaining the Relative humidity (RH) of fermenter at approximately (95-98%) by sprinkling sterile water periodically,

7. Fermenting the entire mixture at 25-30°C by controlling air flow rate (35-40 cc/min)

8. Continuing the fermentation for 14 to 35 days while monitoring the temperature, moisture and Relative Humidity.

In another variation of the present invention, the wood chips containing fragrant resin was sterilized (80° C followed by UV exposure) and fermented with Fusarium by complete soaking. For control 180 g of wood chips was soaked fully in sterile water and placed in the fermenter. For treated, Fusarium suspension prepared from 7-day old PDA plates @ 1.2 X 106 spores/ml was added to 180 g resinous chips. The wood was mixed thoroughly with the suspension and allowed to submerge at 25-30° C in the fermenter for 14-35 days. Example 1: Fungus concentration used to initiate fermentation

Example 2: Quantitative data for Agarwood Oil

Brief description of the drawing

Figure 1 shows the actual photographs of Fermentation of resinous agarwood in by semi-dry method.

Figure 2 shows graphical representation of amount of hydro distilled oil recovered from control and treated resinous agarwood samples fermented in semi dry condition.

Figure 3 depicts a comparison of oil quality with reference to total agarospirol levels obtained from (control and treated) semidry fermented agarwood.

Figure 4 shows the GC-MS data for oil from fermented biomass of the present invention.

Detailed description of drawing:

Figure 1 further explains uninoculated resinous agarwood fermented in water for 7 days (A) and Fungus HI 5 inoculated resinous agarwood fermented in water for 7 days (B) and Re-isolation of fungus from fermented agarwood with fungus HI 5 after 7 days of fermentation. (C) Figure 2 further explains X-axis representing the days of fermentation vs amount of Agarwood oil (g/kg of agarwood) in Y-axis.

Figure 3 further explains blue color bars represent semidry fermentation of control agarwood (untreated) and Brown color bars represent semidry fermentation in presence of fungus (treated)

Figure 4 further explains the chromatogram which confirms the presence of agarospirol in agarwood oil obtained from the fermented biomass of the present invention and showing the peak at 25.7 minutes.

Advantages:

Less time required for the fermentation.

Quantity of oil produced per unit wood consumed (productivity) increased

Quality of oil is enhanced since the agarospirol concentration is increased in lesser amount of time.

Fungus assisted fermentation increases the rate of fermentation as well as has a promising quality of Agarwood Oil.

The current method is cost effective.

The natural process takes 45-90 days for fermentation with the present invention the time is reduced to less than 15 days.

Lesser turnaround time results in quick revenue.