Field of the invention:
The present invention relates to an improved process for extraction of allylisothiocyanate (AITC) from aqueous extract of seeds of Brassica juncea (Indian mustard seed) ) and also for an apparatus for extracting allylisothiocyanate (AITC) from aqueous extract of seeds of Brassica juncea
Background of the invention:
Method of producing allylisothiocyanate by steam distillation has been described in patent number- PR : CS19700007968 The method is based on linear extraction method.
Objects of the invention
The object of the present invention is to overcome the drawbacks of the prior art wherein linear increase in temperature was being used for extraction of AITC from mustard seeds
Yet another object of the present invention is to develop an economic process through water hydrolysis for the extraction of essential oil (allylisothiocyanate)
Still another object object of the present invention is to develop diverse bio-fungicide using allylisothiocyanate, as being the active ingredient and also for pharmaceutical applications as being the anti-carcinogenic compound.
A further object of this invention is to obtain allylisothiocyanate of purity upto 99%.
Yet another object of the present invention is to provide for an apparatus which can be used for the above extraction process
Summary of the Invention
To achieve the afore-mentioned objects, the present invention provides for a process for a process for the extraction of allylisothiocyanate from Indian mustard seeds, the said process comprising steps of
i) cracking the mustard seeds,
ii) extracting the essential oils (AITC) from the cracked seeds of step (i) with water, the temperature of the water being gradually increased from 50° to about 100°
iii) distilling the mixture of step (ii) to obtain a crude distillate,
iv) purification of the crude distillate by condensing the generated vapors of step (iii) by passing through a cooled multi-stage condenser
vi) collecting the condensate,
vii) repeating at least four times steps (iv) and (v) on condensate of step (vi), to obtain allylisothiocyanate of purity 98-99%
The said process utilizes water for hydrolysis instead of steam and a complete extraction and purification assembly for separation and purification of this ingredient. Also, the process has optimized various conditions for extraction of mustard seeds and then arrived at the process for extraction of maximum yield of allylisothiocyanate having enhanced efficiency and with minimum mass loss of active ingredient allylisothiocyanate.
The process is a three-step process for extraction of AITC from mustard seeds.
The present invention also provides an apparatus for also for an apparatus for extracting allylisothiocyanate (AITC) from aqueous extract of seeds of Brassica juncea.
Brief Description Of The Accompanying Drawings or Tables
Figure 1 illustrates process of the present invention
Figure 2 illustrates provides an extraction assembly according to the present invention.
Table-1 provides data on AITC extraction of bold Indian mustard seeds.
Table-2 provides data on AITC extraction of partially crushed/cracked Indian mustard seeds.
Table-3 provides data on AITC extraction of powdered Indian mustard seeds
Table-4 provides extractability percentage of AITC from bold Indian mustard seeds under different conditions.
Table-5 provides extractability percentage of AITC from partially crushed/cracked seeds under different conditions.
Table - 6 provides extractability percentage of AITC from powdered seeds under different conditions.
Detailed Description
Referring to figure 1, the steps of the process include....
Also as shown in figure 2, the apparatus of the present invention comprises
- an incubator/ reactor flask having two openings, one for connecting to air-
space top and other for agitator.
- an air-space top having round shape with three openings, one lower opening for connecting to flask, second side opening for connecting to a condenser and the third top opening for releasing extra pressure or adding water in between the processing and
- a multi-stage condenser having openings at an alternate node on lower side for purification of allylisothiocyanate.
The size of bulb area of this equipment is 1/4th of the size of reactor/incubator flask.
Different cultivars of Indian mustard (Brassica juncea) were used for the extraction of the active ingredient. A specially designed assembly for maximum reaction efficiency and purification of this compound has also been developed.
This invention has been done with multiple replicates adopted for the extraction of allyl-isothiocyanate from crushed mustard seeds. The process of the present invention involves hydrolytic reaction of glucose moiety and a thiosulphate group, which is present in mustard seeds in the form of total glucosinolate, with water in presence of endogenous enzyme called myrosinase.
The break down of total glucosinolate by hydrolysis yield allylisothiocyanate under proper controlled reaction parameters. The crude distillate of the break down product was purified by further three
distillation steps at the boiling point of water. The distillate collected was regularly separated into two parts. First upper layer consists of allyl-isothiocyanate and the lower pungent pigment water layer of milky white to colorless. The upper allylthiocyanate layer was separated continuously by using specially designed assembly with multi-outlets at different levels for proper separation of allylisothiocyanate from milky pungent water received from previous stage.
This active ingredient was analyzed for its fungicidal effects. It was also observed that it works at very high dilutions/low concentrations.
The active ingredient was further utilized for development of a biodegradable diverse bio-fungicide.
The present invention provides a process for the extraction of allylisothiocyanate from Indian mustard seeds, the said process comprising steps of
a) cracking, optionally diluting the washed mustard seeds,
b) extracting the essential oils from cracked seeds of step (a) with water at an initial temperature of 50°-60°, for about 15 minutes, raising the temperature to about 70° and maintaining for 25-30 minutes, further raising the temperature to about 100°c and maintained for about - 60 minutes
c) distilling the mixture of step (b) to obtain a crude distillate,
d) boiling the crude distillate of step (c )
e) condensing the generated vapors of step (d) by passing through a cooled multistage - condenser
f) collecting the condensate,
g) repeating at least four times steps (d) and (e) on condensate of step (f), and obtaining allylisothiocyanate of purity 98-99%
The seed to water ratio ranges between 1: 2 to 4.
The mustard seeds used in the present invention are cracked seeds or partially crushed seeds so as to ensure effective extraction of allylisothiocyanate
Another embodiment of the present invention is to provide for a bio-fungicide composition comprising of an active ingredient allylisothiocyanate.
The complete process of the present invention is divided into following major steps:
Preparation of seeds : Mustard seeds (Brassica juncea) after proper cleaning are crushed partially in a seed cracker. The best cracking for this process is optimized by calculating the cracking percentage of seeds. For better results the seed cracker with 500-700 RPM crushes the mustard seeds in about 10 minutes. The cracking of mustard seeds must be partial and not in powder form. In other words, the seed must break into two or three parts for better enzyme activation to provide enhanced hydrolysis. If seeds are cracked more, it will generate heat and will deactivate myrosinase, which results in less or no production of allylisothiocyanate.
Seed Finishing: After cracking, dehulling of seeds may done for more pure extraction of allylisothiocyanate. However, to save energy consumption dehulling can be avoided. But during the experimental standardization the only difference in both the types of seeds used in the two processes is the presence of colour in allylisothiocyanate product obtained by using seeds with hull. In dehulled seeds the extracted allylisothiocyanate is colourless to pale yellow and in non-dehulled seeds the colour of allylisothiocyanate is brown.
However, there is no other difference in terms of purity or chemical composition..
The seeds after cracking and dehulling are ready for hydrolysis.
Method of Estimating Cracking percentage: 10 gms of seeds were taken from a cracker for at least 10 times and sieved them through different size of sieves. Each category having different size of sieves was weighed.
The following formula was used to work out percentage cracking for different sizes.
Percentage cracking for two piece cracking =
weight of two pieces cracked seedsx100
10
Percentage of all other categories were worked out similarly.
However, the percentage of two piece cracked seeds must exceed 70% for better results.
Reaction Preparation:
Seeds taken from cracker were immersed in a reactor containing water.
Initially the temperature of mixture was maintained between 50-60°C for fifteen minutes and then raised to about 70°C and maintained for about 25-30 minutes.
After this reaction period, the temperature of the mixture was raised to the boiling point and was allowed to boil with continuous agitation.
Volume of water used is two and half times to three times more than the weight of seeds immersed and having a pH of 7- 7.5.
The reactor used to carry out the process includes a stirrer and a rheostatic temperature controller. The stirrer/agitator speed must be between 60-70 rpm to prevent sticking of mustard seeds with walls of cylindrical reactor.
The reactor is connected to a condenser. The condenser is connected at the end with an inverted joint which collects the crude distillate in a receiver tank. This crude distillate is milky white in colour with highly pungent odor. The distillate is a mixture of allylisothiocyanate and water interface partially miscible. This crude distillate is analyzed for its percentage of allylisothiocyanate and this percentage ranges from 0.5 to 1 percent at different stages. This crude distillate is then further boiled and steam is produced. This steam is allowed to go through a long condenser (water-cooled) and further collected. This process is done mostly four times to purify the distillate upto 98-99%
Parameters for condenser
a) Condenser for crude distillate: Length of condenser must always be twenty-four times of its diameter of condensation area (the path from where steam goes and condenses).
b) Condenser for refining crude distillate: Length of condenser must be fifty times of its condensation diameter for efficient condensation, as allyl isothiocyanate is highly volatile.
At the stage, when allylisothiocyanate is partially mixed with crude distillate there are chances of degradation of allylisothiocynate if it is kept for more than two to three days. This also reduces the content of allylisothiocyanate and decreases the yield. It has also been observed that if continuous stirring or agitating is not done during the boiling of mustard seeds in water, charring of mustard produces burning protein odor in the crude distillate/ allylisothiocyanate obtained after the complete extraction.
Once the crude distillate is obtained it is then further allowed to boil by using a water cooled condenser and then the first distillate is collected. Two methods for isolation of pure allylisothiocyanate from water can be adopted:
a) Pungent water receiver
b) Centrifuge separation
Detailed Distillation and Purification Step:
In this process, the mixture of seeds and water is boiled for specified time, the allylisothiocyanate produced is released in the water interface after getting activated by endogenous enzyme myrosinase. This is the major critical step of this process. After getting the release of active ingredient in water it forms an interface and is partially soluble. Now by providing heat the interface is broken down and the essential oil present in water gets evaporated along with water vapours. These vapours are then passed through a standardized/ calibrated (designed on various replicate basis) condenser. This condenser is a long condenser having twenty-four times long of its condensation path's diameter. Water and active ingredient vapours after passing through water cooled condenser cools down to a milky liquid. The milky liquid is then collected in a receiver with small mouth to prevent its loss into the atmosphere. The distillate collected must be 45-50% of the total water used for the extraction. The percentage has been standardized with a series of 50 replicates. The crude distillate contains more than 95% of active ingredient. The left over mass in the reactor having cooked mustard can be directly send to dryer and then to solvent extraction unit for extraction of mustard oil which makes the process of the present invention very much cost effective. Crude distillate collected is then subjected to a series of three to four distillation steps for purifying the crude distillate. The condenser designed in such a way to separate the allylisothiocyanate of different purity level. It has five condensate area in round shape and four of them are having opening in the lower side leaving first one without opening. Through these opening the distillate is collected into different receivers of different purity. This process avoids further purification. When crude distillate is collected at first stage, it is further allowed to boil for 30-35 minutes. From this stage onwards the condenser used must be 50 times long with respect to its inner condensate area (through which vapors are travelling) and the distillate collected must be one-third of the volume of liquid used in the previous stage. The left over water can be discarded off. At this stage distillate collected is purified by two ways one is the gravity action under normal conditions and second is the centrifuge process. However, centrifuge is more appropriate for isolation of this compound from water. During gravity action, the extracted mixture is kept undisturbed for half an hour. There are two layers upper allylisothiocyanate layer and lower drain layer or pungent water layer containing 0.001 to 0.005 percent of allylisothiocyanate and pigments of mustard seed hull. The upper layer is separated and purified by further distillation process. This is pure allylisothiocyanate.
Material and Utility Balance:
The volume of distillate collected at initial stage is half of the volume of water used for boiling of the mixture. After initial stage the volume of distillate collected is 1/3rd of the volume of previous stage distillate. The percentage of
allylisothiocyanate left in the boiling material is analyzed either by smelling it or by HPLC.
However, at this stage HPLC is important for detection of very low quantities of allylisothiocyanate(>5 µ mol/g). However, smelling is important for making it cost effective. Because when there is presence of less than 10µ mol/g allylisothiocyanate in the water phase it will not smell pungent and to extract this low amount of isothiocyanate increases energy cost.
For Basic experimental purposes GLC/HPLC analysis for allylisothiocyanate content in the pungent left over water is very much important for calculation of mass balance.
Mass Balance :
Amount of allylisothiocyanate present in mustard : 0.40% (Average
of 20 replicates)
Processing loss : 2.0%
Net recovery : 0.392%
Extraction Temperature :
a) Incubation temperature (for 15 minutes) : 50°-70°C
b) Reaction Developing temperature (for 30 minutes): 70°-80°C
c) Essential Oil Extraction Temperature (1.5hours) : 80°-100°C Extraction efficiency :
Reaction efficiency for these standardized parameters is more than 95% and mass balance has a loss of 2%(processing loss). However, this is the most efficient reaction efficiency and mass balance for such a volatile compound. The percentage of allylisothiocyanate as Glucosinolate present in mustard seed ranges from 0.4 to 0.45% depending upon the cultivar to be used.
Separation procedure:
At each distillation step from second distillation onwards two types of methodologies can be adopted:
a) Pungent water receiver
b) Centrifuge separation
Pungent water receiver: Pungent water receiver has been designed particularly for isolation of allyl-isothiocyanate from water phase. As allyl-isothiocyanate is lighter than water therefore the opening of pungent water receiver has been developed at the top below the neck. The length of the pungent water receiver,
maintained at room temperature, must be four times its width for proper separation. This separator acts under gravity action and saves energy cost.
After receiving the distillate from pungent water receiver, it is then subjected to multi stage condenser for enhancing its purity with minimum moisture level. In multi stage condenser there are 4-5 condensing areas for collecting the distillate The distillate is collected in pungent water receiver for further separation which yields pure allylisothiocyanate. The purity enhances with the number of the distillation/condensation or more relevant to say with respect to length of the condenser.
Centrifuge Separation: Centrifuge is the already available method for separation of two different density liquids. In the present process centrifuge separation method involved consumption of more energy. Hence pungent water receiver method is preferred over centrifuge separation.
The present invention will now be described with reference to the fore-going examples
Example 1:
For extraction of allylisothiocyanate from mustard seeds various replicates and methods has been adopted during this experiment. Some worked very well whereas some had given negative results.
For example when intact bold mustard seeds were used for extraction of allylisothiocyanate the extraction of the principle ingredient was between 6-12.4%. Increase from 6-12.4% was due to increase in the amount of water ratio from 1:1 to 1:4 with already standardized reaction parameters (see table 4-6). The standardized reaction parameters are incubation of seed and water mixture for 15 minutes at 50-60°C then raising the temperature to 70-80°C and maintained for 30 minutes then the temperature is raised to boiling 80-100°C for about 1.5 hours. The highest extraction percentage is 12%. (Table 1)
Example-2:
In second case seeds used were partially crushed. Partially crushed means breaking of one seed into 3-5 pieces with particle size of 0.15-0.5mm. In this case the seed/water ratio was the same as from 1:1 to 1:4 and the temperature reaction parameters were also the same. The extraction percentage of allylisothiocyanate in this case ranges form 45-97%. (Table 2)
Example -3:
In third case seeds used were in powdered form. In this case the seed/water ratio was more as compared to other two cases because it was observed that powdered seeds absorb more water as compared to other two categories the seed ware ratio is 1:1 to 1:5. The extraction percentage of
allylisothiocyanate in this category was between 62-93.7%, keeping all the reaction temperature conditions the same (Table 3)
Therefore, it is evident from the above examples the partial crushed seeds are ideal for extraction with the said standardized reaction parameters.
The present invention is not to be limited in scope by the specific embodiments and examples which are intended as illustrations of a number of aspects of the invention and any embodiments which are functionally equivalent are within the scope of this invention. Those skilled in the art will know, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

We claim:
1. A process for the extraction of allylisothiocyanate from Indian mustard
seeds, the said process comprising steps of
i) cracking the mustard seeds,
ii) extracting the essential oils from cracked seeds of step (i) with water, the temperature of the water being gradually increased from 50 ° to about 100°
iii) distilling the mixture of step (ii) to obtain a crude distillate,
iv) boiling after dilution with water the crude distillate of step iii
v) condensing the generated vapors of step (iv) by passing through a multi stage cooled condenser
vi) collecting the condensate,
vii) repeating at least four times steps (iv) and (v) on condensate of step (vi), to obtain allylisothiocyanate of purity 98-99%
2. A process as claimed in claim 1 wherein the seed to water ratio ranges between 1: 2 to 4.
3. A process as claimed in claim 1 wherein the mustard seeds are cracked seeds or partially crushed seeds so as to ensure effective extraction of allylisothiocyanate
4. A process as claimed in claim 1 wherein in step ii, the initial temperature of water temperature is maintained at 50°-60°, for about 15 minutes, thereafter, raising the temperature to about 70° and maintaining for 25-30 minutes and then further raising the temperature to about 100°c and maintained for about 60 to 90 minutes
5. An apparatus for extracting allylisothiocyanate (AITC) from aqueous extract of seeds of Brassica juncea comprising
- an incubator/ reactor flask having two openings, one for connecting to
air space top and other for agitator, -an air-space top having round shape with three openings, one lower opening for connecting to flask, second side opening for connecting to a condenser and the third top opening for releasing extra pressure or adding water in between the processing and
a multi-stage condenser having openings at an alternate node on lower side for purification of allylisothiocyanate.