DESC:FIELD OF THE INVENTION
The invention relates to an enzyme preparation for extraction of palm oil. The invention also relates to a process of extraction of palm oil using the enzyme preparation.

BACKGROUND OF THE INVENTION
Palm fruit is a rich source of oil. Trees bearing palm fruits are perennial and have economic life of about 25 years. However, the growing demand for palm oil faces impediments such as, scarcity of land bank for cultivation, labour intensive dependency and negative environment implications. These weaknesses are a concern and hotly debated around the world. The traditional process for the extraction of oil from palm fruit mesocarp leaves substantial amount of oil in the cake, which is not recovered and goes waste.

Enzyme assisted oil extraction processes have been described and claimed to increase the oil yield from the palm fruits with lesser oil wasted in the cake. All prior art essentially describe the usage of majorly cellulolytic enzymes, such as cellulases, cellobiohydrolases, along with other enzymes including various hemicellulases as a minor component (Rathi et al., 2012; Eshtiaghi et al., 2015; Silvamany et al.,2015; Guha et al., 2015; Borch et al; 2014; Guha et al., 2016).

Further, palm oil extraction is a heat dependent process. The process temperature ranges from 70 to 90°C. Prior art describes the use of traditionally known enzyme preparations for palm oil processing which operate over a wide temperature range. However, it is well known that most of these enzymes get inactivated when exposed to such high temperatures for a longer period. Various methods or processes have been proposed and used to overcome the problems associated with loss of enzyme activity at such high temperatures.

WO2012011130 describes lowering the process temperature below 65°C to suit the multi enzyme preparation. But, it has been observed that at lower process temperatures the Mass Passing to Digester (MPD) becomes thick, viscous and difficult to handle, thereby affecting the production and throughput. Secondly, due to the absence of heat tolerant enzymes in this preparation, the oil yield obtained is not so significantly enhanced, affecting techno-commercial feasibility.

There have been disclosures related to improving crude palm oil yield using an enzyme which degrades a phospholipid present in said palm fruit or portion thereof or palm fruit extract; and for process for extraction of crude palm oil from palm fruitlets comprising contacting the palm fruitlets with an enzyme preparation comprising cellulose at temperature of above 65°C.

However, there still exists a need for an enzyme preparation which is optimally active at palm oil extraction process temperature i.e. in the range of 70 to 95°C, more specifically 80 to 95°C and also ensures a higher recovery of oil from the palm fruit.
SUMMMARY OF THE INVENTION
The present invention in one embodiment discloses an enzyme preparation which works optimally at process temperatures mentioned and gives improved oil extraction. The enzyme preparation comprises at least one thermostable enzyme that catalyses the hydrolysis of pentosans, more specifically mannans and hetero-mannans.

Typically, the thermostable enzyme used in the present invention retains more than 50% activity at the process temperature of 70°C and above.
With the use of this thermostable enzyme, the oil yield is observed in the range of 1-20%

In another embodiment of the present invention, the enzyme preparation further comprises at least one enzyme selected from the hydrolase class of enzymes. The said hydrolase class can be selected from carbohydrase group of enzymes. Furthermore the carbohydrases can be selected from one or more of the following enzymes: cellulases, cellobiase, beta glucanase, xyalanse, amylase, xylosidase, pectinases, and/or beta glucosidase.

The preparation containing thermostable mannanase and any/all of the enzymes from hydrolase class of enzymes gives an oil yield in the range of 1.5-25%

The present invention in another embodiment discloses a process for enhanced extraction of palm oil using the enzyme preparation.

DEFINITIONS
The term ‘thermostable enzyme’ as used herein means an enzyme which maintains at least 50% of its unit activity in and at process temperatures of above 70°C for a time range of 1-30 mins of incubation where one unit activity is defined as 1 micromoles of reducing carbohydrates released per minute.

The term ‘hydrolysis’ as used herein means the breakdown of complex carbohydrate moieties present in the palm mesocarp.

The term ‘pentosans’ as used herein means group of polysaccharides found with cellulose in palm mesocarp which under enzymatic reaction yields smaller polysaccharides and/ or pentose sugars on hydrolysis.

The term ‘mannans and hetero-mannans’ as used herein means plant polysaccaharies containing linear polymer of mannose and having beta (1-4 linkages)

The term ‘hydrolase class of enzymes’ as used herein means the enzymes which are capable of hydrolysing complex carbohydrate moieties present in the palm mesocarp which hydrolyse complex carbohydrates to release lower molecular weight polysaccharides and/or mononers

The term ‘carbohydrases’ as used herein means enzymes that break down carbohydrates and fibres to oligosaccharides and/or simple sugars.

The term ‘cellulases” as used herein refers to an enzyme that hydrolyses a material containing cellulose. The total cellulolytic activity may be determined by hydrolysis of carboxymethyl cellulose (CMC) at pH 4.5 and at 40° C, and the resulting reducing sugars quantified by the DNS method . One enzyme unit is defined as the amount of enzyme releasing 1 µmol reducing sugar, per gm or ml, per minute under the conditions of assay.

The term ‘cellobiase’, as used herein refers to an enzyme which hydrolysis the ß-glucosidic linkage of a cellobiose molecule to give two molecules of ß-D-glucose. The cellobiase activity is expressed as the number of enzyme units per ml of crude cellulase at pH 4.8, Temp 50°C with cellobiose as substrate. One enzyme unit is defined as that quantity of enzyme which produces 2 pmol glucose/min under the conditions of the assay.

The term ‘beta glucanase’ as used herein refers to an enzyme that hydrolyses ß-glucans. The beta glucanase activity is determined by hydrolysis of ß-D-glucan from barley at 30°C, pH 5.0. One enzyme unit is defined as that quantity of enzyme which liberates 1 µmole of reducing sugars in one minutes under the conditions of the assay.

The term ‘Xylanase’ as used herein refers to an enzyme that hydrolyses xylan containing material. The xylanase activity is determined by hydrolysis of Beechwood xylan at 50°C, pH 5.3. One enzyme unit is defined as the amount of the enzyme which liberates 1 µmol of reducing sugar measured as xylose equivalent from xylan per minute under the conditions of the assay.

The term ‘Amylase’ as used herein refers to an enzyme that hydrolyses glucosidic bonds present in starch to liberate sugars. The amylase activity is determined by hydrolysis of potato starch at 30°C, pH 6.6. One enzyme unit is defined as the quantity of enzyme that will dextrinize starch at the rate of 1 mg/min under the conditions of the assay.

The term ‘xylosidase’ as used herein refers to an enzyme that catalyses the hydrolysis of terminal, non-reducing alpha-xylose residues in alpha-xylosides. One enzyme unit is defined as the amount of the enzyme catalysing the hydrolysis of 1 µmol of substrate (maltose) in 1 min at 65°C at pH 5.50.

The term ‘beta glucosidase’ as used herein refers to an enzyme that catalyzes the hydrolysis of terminal non-reducing residues in ß-glucosides, with release of glucose. The beta glucosidase activity is determined by hydrolysis of D-Salicin at pH 4.8, Temperature 50°C. One enzyme unit is defined as the quantity of enzyme that liberates 1 µmole of sugars in one minute under the conditions of the assay.
The term ‘mannanase’ as used herein refers to an enzyme that catalyzes the hydrolysis of mannans. The mannanase activity is determined by hydrolysis of locust bean gum at pH 5.3 and at 50° C, and the resulting reducing sugars are determined by the DNSA method. One mannanase unit is defined as the amount of enzyme that produces reducing carbohydrates having a reducing power corresponding to one micromole mannose from mannan in one second under the condition of assay.

The term ‘mesocarp’ as used herein means middle layer of the pericarp of palm fruit, between the endocarp and the exocarp, on which the enzyme acts.

The term ‘mass passing digester’ as used herein implies to the stripped palm fruits after sterilization and before it enters the digester.

BRIEF DESCRIPTION OF THE DRAWINGS
The above mentioned, other features and advantages of the various embodiments of the invention, and the manner of attaining them, will become more apparent and will be better understood by reference to the accompanying drawings, wherein:

The figures show microscopic images of effect of enzymes on the lipid bodies present in the palm mesocarp.
Figs 1a, 1b and 1c depicts the control without any enzyme treatment
Figs 2a, 2b and 2c depicts the action of thermostable enzyme
Figs 3a, 3b and 3c depicts the action of cellulase
Figs 4a, 4b and 4c depicts the combined action of thermostable enzyme and cellulase

DETAILED DESCRIPTION OF THE INVENTION
In order to find an improved enzymatic solution for oil extraction in the given process conditions, i.e., at the elevated temperatures, the present inventors have observed significant increase in the efficiency of palm oil extraction using a thermostable enzyme preparation.

The thermostable enzyme preparation of the present invention catalyses the hydrolysis of pentosans, more specifically mannans and hetero-mannans.
Preferably, the thermostable enzyme disclosed in the various embodiments is a thermostable mannanase.

Mannanase has been used as a feed additive enzyme to increase the nutritional value of major components of animal feed such as soybean meal, copra cake, palm kernel meal and guar meal [Lee et al., 2005; Wu et al., 2005;Yoon et al., 2008] and laundry detergents [McCoy, 2001; Schafer et al., 2002]. Mannanase has also been reported for the conversion of biomass substrates comprising mannans such as coffee waste, guar meal, palm kernel cake, palm kernel meal and/or copra cake into a fermentation product (Soerenson et al., 2007). Therefore it comes to light, that although mannanases have been used in processing of substrates with mannan content, an enzyme preparation containing primarily mannanase or centered around mannanase have never been reported to increase oil yield during palm oil extraction.

Mannanase of the present invention is capable of acting on the mannans and heteromannans present in the mesocarp of the palm fruit. Mannans and hetero-mannans although present in small quantities in the palm fruit mesocarp (Saka et al., 2008), appear to contribute significantly to the integrity of palm oil bearing bodies. This becomes quite evident from the increase in oil yield obtained by the use of mannanase containing enzyme preparation as elucidated in the present invention. The enzyme preparation of the present invention selectively ruptures the cell membrane of the oil bearing bodies. Consequently, the loosened cell well matrix releases more oil on application of pressure. This finding is corroborated in Figs 2a to 2c. Mannanse treated mesocarp tissue compared to enzyme untreated mesocarp tissue is observed to have reduced oil content.

The thermostable mannanase of the present invention retains more than 50% activity above 70°C.
Furthermore, the thermostable mannanase at 70°C retains activity in the range of 90-95% for 10 mins, activity in the range of 80-85% for 20 mins and activity in the range of 60-65% for 30 mins. The thermostable mannanase at 80°C retains activity in the range of 90-95% for 10 mins, activity in the range of 70-75% for 20 mins and activity in the range of 50-55% for 30 mins. The thermostable mannanase at 90°C retains activity in the range of 10-15% for 10 mins and activity in the range of 5-10% for 20 mins. The thermostable mannanase at 95°C retains activity in the range of 1-5% for 5 mins.

Further, it has been observed that the enzyme preparation used for extraction of palm oil is used in the range of 0.5-200units/gram, more specifically 1-100units/gms, more specifically 5-80units/gms or more specifically 10-40units/gms. The oil yield is increased in the range of 1-20% using said enzyme preparation.

In another embodiment of the present invention, the preparation further comprises at least one enzyme selected from the hydrolase class of enzymes.

In an embodiment, the hyrolases class can be selected from carbohydrases such as cellulases, cellobiase, beta glucanase, xyalanse, amylase, xylosidase, pectinase, beta glucosidase and/or combination thereof.

The enzyme preparation comprising the further enzyme gives increased oil yield in the range of 1.5-25%.

In another exemplary embodiment of the present invention, the preparation may further comprise of suitable diluents and stabilizers.

The present invention in another aspect discloses a process for enhanced extraction of palm oil using the enzyme preparation. In one aspect, the present invention relates to a process for extraction of palm oil from palm fruits involving steps of contacting the palm fruits with the enzyme preparation comprising at least one thermostable enzyme that catalyses the hydrolysis of pentosans, more specifically mannans and hetero-mannans at a temperature of above 70 °C and extracting the crude palm oil. The process involves initially stripping and sterilizing the palm fruits followed by adding an enzyme preparation comprising mannanase to the palm fruit and holding at a temperature ranging from 70 to 90 ?C for a contact time in the range of 5 to 60 minutes. Further pressure is applied and the oil is collected.

In an exemplary embodiment of the present invention, the palm fruits are stripped and steam heated (referred to as ‘sterilization’ in the palm oil industrial practise). These sterilised fruits are then slightly pressed/mashed to obtain the pressed/mashed palm fruit mass. These are then conveyed into the digester. The enzyme preparation is sprayed/sprinkled in predetermined amounts onto the conveyer loaded palm fruit mass during conveyance. Optionally additional predetermined amount of the enzyme preparation is added in the digester followed by through mixing. This mixture is further contacted/incubated for 5 to 60 minutes at 70 to 90 ?C to obtain a digested mass. Further pressure is then applied to the digested mass to obtain crude palm oil which is further processed to obtain the palm oil.

In the typical example, the enzyme preparation is sprinkled or sprayed onto the palm fruit mass during conveyance to the digester, which leads to improved exposure of the palm fruit mass to the enzyme preparation due to increased contact time between the enzyme preparation and the fruit, thereby leading to enhanced palm oil extraction.

Typically, the digested mass is passed into a screw press, from which crude palm oil is obtained.

Accordingly, while this invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications of the illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to this description. It is therefore contemplated that the appended claims will cover any such modifications or embodiments as fall within the true scope of the invention.

EXAMPLES:
Example 1: Effect of enzyme treatment on the lipid bodies present in the palm mesocarp
Palm fruits used in the present invention were obtained from Malaysia.
The palm mesocarp was finely sectioned and rinsed with hexane. The sections were subjected to enzyme treatment followed by microscopy studies to evaluate effect on the lipid bodies. Mesocarp sections were treated with Thermostable mannanase, cellulase and a combination of both thermostable mannanase and cellulase at a temperature of 90°C for 30 min. Control sections were subjected to treatment with temperature of 90°C for 30 min with addition of water only. Thereafter, the sections were fixed and stained with Sudan black B for lipid staining. The stained sections were then observed by light microscopy (Olympus, BX 43) at 100X to examine the lipid bodies in the mesocarp

Inference: The mesocarp parenchyma cells of oil palm are fully packed with lipid bodies. Sudan black staining method was used for the staining of lipids in the mesocarp. The lipid bodies in the mesocarp, stained by the dye appear black. The control cells without enzyme treatment, show dark globular masses within the cells (Figs 1a to 1c). The enzyme treated cells show, a varied degree of reduction in the lipids, as obvious from reduction in staining. This shows that enzyme-treatment is positively influencing extraction of lipids from the mesocarp cells. Mannanase and cellulase when used singly, show small droplet-like lipid structures within the cells at a much lower distribution compared to the control (Figs 2a to 2c and Figs 3a to 3c). However, treatment with both enzymes together (Figs 4a to 4c), shows a marked reduction in lipids. In this case, many cells even appear devoid of lipid bodies, suggesting significant lipid extraction.

Example 2: Effect of enzymes on palm oil extraction
Lab scale trial
2kg palm fruits were steam treated at 110oC for 90mins. Sterilized fruits were further thrashed in spiral mixer. The mesocarp and palm kernels were separated. 100g of thrashed mesocarp was subsampled for enzyme treatment and incubated at 90oC. Enzyme samples at 500ppm dosage were diluted with 10gm water and mixed with palm mesocarp. Reaction was carried out in mashing bath for 30mins at 90oC. Post reaction, samples were cooled and subjected to extraction with 100ml n-hexane. This n-hexane fraction containing oil was evaporated in rotatory evaporator and weight of the oil was measured.
Control sample was treated with same protocol with no enzyme addition.
Table 1
Sample Enzyme dosage (ppm) Oil collected (gms) Oil Extracted (%)
Control 0 42 100.00
Enzyme 1 500 46 109.52
Enzyme 2 500 48 114.29
Enzyme 3 500 52 123.81

Results: Table 1 below illustrates the effect of enzyme preparations on oil extractions as compared with the control with no enzyme:
Control: No enzyme
Sample 1: Only Thermostable Mannanase (10,000 – 25,000 units/g)
Sample 2: Cellulase (1000-7000 units/g) having side activities such B-glucanase and xylanase
Sample 3: Thermostable Mannanase (10,000 – 25,000 units/g) Cellulase (1000-7000 units/g) having side activities such B-glucanase and xylanase
Scale up Trial:

Initially 100Kg mass passing digester (MPD) was collected as a lot from a operating mill in Malaysia and 10kg MPD was sub sampled for each experiment. Further enzyme sample with varying dosage (as depicted in table 1) was mixed in 1Kg water (10%) and applied on 10kg MPD. The enzyme applied MPD was then added in a digester where digestion reaction was carried out at 90 ?C for 30 minutes. Post 30 minutes, the digested reaction mass was pressed in a screw press from where the press liquor and fibres were obtained. The press liquor was further diluted with dilution water and overall liquid volume was adjusted to 10 litres.
This diluted liquor was further heated to 90 ?C using steam and was uniformly mixed. The mixed liquor was subjected to centrifuge spin test at 5000g for 5 min to obtain oil, water and sediment concentrations.
Results: Table 2 below illustrates the effect of enzyme preparations on oil extractions as compared with the control with no enzyme:
Control: No enzyme
Sample 1: Only Thermostable Mannanase (10,000 – 25,000 units/g)
Sample 2 : Cellulase (1000-7000 units/g) having side activities such B-glucanase and xylanase
Sample 3: Thermostable Mannanase (10,000 – 25,000 units/g) + Cellulase (1000-7000 units/g) having side activities such B-glucanase and xylanase

Sample Enzyme
Dosage
(PPM) Volume
Collected
(ml) Dilution
added
(ml) Oil % Water
% Sediment
% Oil in
Fiber
%
Control 1 0 6100 3900 22 49 29 5.24
Enzyme
1
500
8500
1500
23%
51
26.5
5.14
Enzyme
2

500

8000

2000

24.5%

46

29

5.53
Enzyme
3

500

9000

1000

27.5%

43

29.7

4.69
Table 2: Effect of enzyme on oil extraction

With respect to both the examples, enzyme 1 comprising thermostable mannanase shows a noticeable increase in the oil yield, as compared to the control, which is the crux of the invention. Heteromannans, is as miniscule component in the palm fruit mesocarp. Yet the data presented in example 1 and 2 and Figs 2a to 2c, exhibits the effect of mannanase on palm oil extraction. We believe that the mannanase preparation of the present invention selectively affects the cell wall integrity of the oil bearing bodies. Consequently, the loosened cell well matrix releases more oil on applying pressure.
Enzyme 2 comprising mainly cellulase showed slightly better result than with enzyme 1.
The enzyme preparation of Enzyme 3 comprising thermostable mannanase and cellulase showed the best oil yield as compared to Enzyme 1 and 2. This shows that the further addition of the hydrolases enzymes such as cellulases, pectinase, beta glucannase, xylanases and/or amylase to the thermostable mannanase, results in increased palm oil extraction yield. Synergistic action of thermostable mannanase and cellulase is thereby indicated.

Plant Scale Trial:
A plant trial was conducted in a 60 TPH oil palm mill, with a sample size of 3600 tons fresh fruit bunch (FFB). In the plant, FFB was sterilized and followed by thrashing. Mass passing digester (MPD) received post thrashing was added in digesters continuously followed by pressing. Enzyme 1 (a mannanase enzyme preparation) was added at the top of digester continuously at the dosage of 500ppm per ton of FFB by suitable dilution with water with respect to FFB. Residence time for the enzymatic reaction was adjusted to 15min at 85 to 90°C in a digester. Reacted mass was pressed using double screw press for oil recovery. Pressed oil was further recovered in clarification and oil recovery section. Recovered oil was collected in crude oil tank and was measured using dip method. Control batch was carried out using same methodology as mentioned above without dosing enzyme.
Results: Table 3 below illustrates the effect of enzyme preparations on oil extractions as compared with the control with no enzyme:
Control: No enzyme
Sample 1: Only Thermostable Mannanase (10,000 – 25,000 units/g)

Sample FFB processed (Ton) Oil Recovered (Ton) Oil Recovery (%)
Control 3604.715 592.165 100
Enzyme 1 3600 606.082 102.5
Table 3: Effect of enzyme on oil extraction

It is evident from Table 3 above that in a commercial plant; thermostable manannase enzyme preparation gives a 2.5% increased oil recovery as compared to the control.

The technical advancements offered by the present disclosure include the realization of:
? an enzyme preparation for enhanced recovery of oil from the palm fruit; and
? an economic and simple enzymatic process ensuring a higher recovery of oil from the palm fruit.

Alternations and modifications of the embodiment described herein will become apparent to those skilled in the art. Therefore the scope of this invention should not be unduly restricted by this description which should only be limited by the appended claims.

While the invention has been described with a certain degree of particularity, it is manifest that many changes may be made in the details of construction and the arrangement of components without departing from the spirit and scope of this disclosure. It is understood that the invention is not limited to the embodiments set forth herein for purposes of exemplification, but is to be limited only by the scope of the attached claim or claims, including the full range of equivalency to which each element thereof is entitled.
,CLAIMS:WE CLAIM:
1.) An enzyme preparation for extraction of palm oil, said preparation comprising:
? at least one thermostable enzyme that catalyses the hydrolysis of pentosans, more specifically mannans and hetero-mannans.
2.) The preparation as claimed in claim 1, wherein said thermostable enzyme retains more than 50% activity above 70°C.
3.) The preparation as claimed in claim 1, can be used effectively used for extracting palm oil in the range of 0.5-200units/gram, more specifically 1-100units/gms, more specifically 5-80units/gms or more specifically 10-40units/gms
4.) The preparation as claimed in claim 1, wherein oil yield increase is in the range of 1-20%.
5.) The preparation as claimed in claim 1, wherein said preparation further comprises at least one enzyme selected from hydrolase class.
6.) The preparation as claimed in claim 6, wherein said at least one enzyme selected from the hydrolase class of enzymes is selected from carbohydrases.
7.) The preparation as claimed in claim 7, wherein the carbohydrases are selected from cellulases, cellobiase, beta glucanase, xyalanse, amylase, xylosidase, beta glucosidase and/or combination thereof.
8.) The preparation as claimed in claim 5, wherein oil yield increase is in the range of 1.5-25%
9.) A process for enhanced extraction of palm oil using an enzyme preparation, said process comprising of:
stripping and sterilizing palm fruits followed by contacting said palm fruits or said palm fruit with an enzyme preparation comprising at least one thermostable enzyme that catalyses the hydrolysis of pentosans, more specifically mannans and hetero-mannans, at a temperature in the range of 70 to 90 °C for a time duration in the range of 5 to 60 minutes followed by extracting said palm oil by applying pressure.
10.) The process as claimed in claim 9, wherein said enzyme preparation is sprayed/sprinkled on said palm fruits or said palm fruit portions during conveyance to the digester or in the digester itself.