There are several existing methods for the extraction of oil, such as solvent extraction method and aqueous extraction method. These methods are not economic and need more time for the extraction of oil. My project demonstrates the use of biotechnology to extract edible oil from the seeds of mustard. The study on the enzymatic extraction of oil from the mustard found that the best ratio of cellulase and pectinase for the optimum extraction of oil from the mustard seeds was 1:2 of cellulase: Pectinase and the concentration of enzymes used were 0.1 Percentage of pectinase and cellulase. The temperature used was about 50 degree celsius and 1 hour of heating time. The best oil yield was about 10.22 percent and this also yielded high protein content of about 25.7 percent. All the parameters effected the oil and protein production significantly except the moisture. The result obtained concluded that the use of enzymes for the extraction of oil using 0.1 percent of enzyme concentration and 1:2 ratio of enzymes was more economic and more productive than the other existing methods such as Aqueous and solvent extraction methods.
The oil industry in india is the most vital contribution towards the development of indian economy. A more than 20 million hectares of the land is used used for growing vital oil seed crops. Although there is a cultivation of oil crops in india over more than 20 million hectares, still there is gap between supply and demand. That is the demand is very much high than the supply of oil. The more stressing topic is that this gap is not narrowing its widening. To overcome this problem there is a need for improvements in yield, post-harvest processing and value addition are very much vital for increasing oil production with better quality oil and to contribute towards indian economy. The human diet is highly depended on the consumption of over more than 90 percent of the world oil production from various sources such as vegetables, animals and marine bio sources. The oil is also used as a culinary element. As it is a fact that breakdown of fats releases two times the energy produced or generated by the equal amount of energy when there is a sugar breakdown. The fattyacids which are present in the oil are very important and play a vital role in nutritional value. The fatty acids that are present in oil also contribute to aromatic nature and are also used as flavor contributors and palatability enhancers along with the carriers of fat soluble vitamins.
Our day to day life is dependent on oil in various ways and in various roles. The edible oil in India is an vital part of palate since ancestral days. In this context India is the largest reservoir for different types of edible oil for both production and consumption of these oils. There is also a particular choice of oil for different states. That is the type of edible oil in India differs from state to state. For example, In the west side of India groundnut oil or the cottonseed is most preferred. In the north-side the people prefer mustard oil over other oils. In Tamilnadu they prefer coconut oil. The production of these oils is very well established and there is a defined and organized process for the production of these oils. Here we are only focused on the production and its defined process which leads to the production edible mustard oil. The process starts from the point where the dry mustard seeds are crushed in a table Ghani, this process can be done using another extractor and using Ghani 90 percent oil can be extracted. There are filters in the industry which are used for the isolation of liquid portion from the solid portion. The solid portion which is separated is called as oil cake and can be used widely as a cattle fodder. The edible oil is then sent to packing where the oil is packed into packets, tins and plastic jars which are approved and of food grade. This packing process can be manual or automated. In automated process, the machines are used for the packing purposes. The produced oil quality is dependent on the quality of seed but averagely if it is seen, the oil produced is about 30 percent to 40 percent of the seeds used in the production. For the commercialization and marketing of the oil produced needs high investment of money. That is capital investment is also a factor that influences the sales percentage of the oil produced. The extraction plant also needs high capital cost along with transport cost. If we take an assumption if for buying the land it costs 0.5 lacs then for building or setting up an oil extraction plant in that land would cost 1.75 lacs. By keeping in mind the demand of the oil and its contribution towards the economy, it is advisable to set up an extraction plant which can produce up to 72 tons of edible mustard oil every year at an efficiency of 100 percent. As we know there would be a requirement of manual labor are also for the production of oil and also there must be thorough qualitative analysis of the oil before it is sent to market for selling. This is done because poor quality of oil may need to health problems. So the quality assurance and quality control department is vital for the assessment of oil quality.
A quality control determines the standard factors that are essential and reviews the all factors quality that are involved in the production of oil. There are a many of inspection factors starting from visual inspection to microscopic inspection. This is done to obtain good detail of product before sending it to the market for sales. These inspectors are provided with a series of inspection factors such as product defects, cracks or surface blemishes that may prove to be not acceptable for consumption.
This quality control departments sends their report to lab and there it is decided to allow or not to allow the product. whereas quality assurance department tries to avoid or tries to reduce the issues with the product and make it acceptable for consumption.
There are a many of factors that are inspected by quality control such as moisture content, refractive index, specific gravity,acid value,pungency,iodine value,saponification,unsaponificable matter so that the edibility quotient or standard is maintained.
So as there is a high expenditure for the production of oil that also include setting up the plant and buying machinery. To reduce the cost many chemical and biological methods for extraction of oil are being researched. The existing methods are solvent extraction method, aqueous extraction method. There is also a need for refining of oil. The use of biotechnology for the extraction of oil would reduce the cost of extraction. So the enzymatic methods for the extraction of oil are being researched. Degumming methods are also being researched for the refining of oil.
2.0 Aims and Objective:
- Extraction of oil using different methods of oil extraction
- Extracted oil Qualitative analysis.
- Extracted oil quantitative analysis.
- Fatty acid profile of oil..
- Oil degumming.
- Extraction of protein as a byproduct
- Quantitative analysis of protein as a byproduct.
3.0 Review of Literature
The experimentation on Mustard was started from the age of romans. To make something new known as burning must they mixed the grape juice which is unfermented with the mustard seeds which were ground.
Mustard seeds were also exported to other places such as Gaul but by the 10th century the monks who belonged to St. Germain of Paris explored the mustard making knowledge which belonged to romans and started their own production pattern. By 13th century a place named Dijon of France became a recognized center for the production of mustard. Then by the partnership of Grey-Poupon and Maurice Grey a unique variety of mustard, the aromatic mustard was produces using white wine.
Mustard belong to the family brassica, the word mustard was derived from Latin which means burning must. During its first utilization it was used more as an medicinal plant. It was considered as a medicine for scorpion stings.
The Mustard Gas, a very dangerous and lethal weapon is an artificial gas made by using volatile nature of oil derived from mustard.
In some diseases the free radicals that are produced in the humans due to metabolism, these free radicals produced may cause oxidative damage. This oxidative damage may be harmful for many molecules. These are also involved in initiation phase of many diseases which are degenerative and and which are malignant. The factors such as lack of good diet, intake of tobacco or smoking of tobacco are the factors that are involved in the cancers that occur in lungs, colon and bladder. The antioxidants are very much vital for the removal of oxidative free radicals to prevent damage, these antioxidants are produced or the excellent sources for these antioxidants are leafy vegetables, nuts and some of the vegetable oils.(Weisburger, 1991). Anticarcinogenic activity is also present in antioxidants. This was seen in the antioxidants produced by crucifers(Kurlich et al, 1999). The chronic diseases can be prevented by consumption of the crucifers(Carol et al, 1996). Studies and experimentation on crucifers provided with a result that concluded that there is an inverse proportionality between consumption of crucifers and development of cancer. The glucosinolate which are a class of phytochemicals are known to be present in crucifers. The anionic compounds such as Sulphur, nitrogen and glucose group are main or vital compounds that are present in glucosinolates( Kjaer, 1960;Ettlinger and Kjaer,1968; Sorenson,1996). The glucosinolates were found to be a major component in almost all the parts of plant. There was also a founding that almost about 15 types of different glucosinolates were found in the same plant. The study on the amount variation in different parts of plant found that according to the age of the part and health of the given plant and the nutrition uptake of the plant the glucosinolate amount varied (Sang et al, 1984; Clossais- Besnard and Larher,1991;kirkegaard and Sarwar,1998). The study on the glucosinolate presented with an interesting finding that the families which contain glucosinolates also contain an enzyme myrosinase that is a thioglucosideglucohydrolase(Buchwaldtetal,1986;Bjergegaard et al,1994; Bones and Rossiter,1996;Bjergegaard et al,2003;Bellostas et al,2003;Petersen et al,2003). There is a study that also found that when a plant tissue is damaged, the glucosinolates come into contact with myrosinase which is a thoglucosidegluhydrolase there is a hydrolytic reaction which leads to the formation of hydrolytic products of the glucosinolates. Further studies on Myrosinase revealed that myrosinase is not a single enzyme but a class of enzymes or group of enzymes or a family of enzymes. A study on the enzymatic reaction of the myrosinase on the glucosinolate as a substrate found that the glucossinlates were broken down into isothiocyanates, epithionitriles, nitriles and thiocyanates. The further studies on these broken down products obtained from the glucosinolate breakdown had varied biological activities such as biocidal(Vig et al,2010) and anticarcinogenic activity(Rosea et al.2005), antimutagenic activity(Rampal et al,2010), antioxidant activity(Barillari et al,2005). They provide us with a perfect demonstration of the importance of the secondary plant molecules or products in the context of plant relationship with pests. The products produced by the breakdown of glucosinolates using myrosinase are bioactive and possess capability and effectiveness to be used as a natural pesticide for pest control purposes. This pest control purpose comprises their action against fungi, nematodes and bacteria(Kirkgaard and sarwar,1998;Smolinska et al,2003;Kirkegaard and matthiessen,2004;poulsen et al,2008). A study found that the breakdown products of glucosinolates were more safer biofumigants in the control of pests. This was because they were biologically degradable and toxicity effect was less compared to chemical pesticides(Vig et al,2009).
4.0 INDUSTRIAL VISIT:
As we know every industry contains machinery to produce their products. In Puri oil mills limited it is no different they have two units. One is for the production of oil and another one is protein extraction unit. The machinery used for the extraction of oil in Puri oil mills limited is cold mill or cold press. In cold pressing machinery or in the milling machinery crushing of the mustard seeds is done. In this cold press or cold mill process there is no use of extra heat or chemicals. It is natural and only cold press is used mechanically or hydraulically pressing or crushing the seeds to extract oil. For every 10 degree Celsius increase in temperature the chemical reaction becomes two times. The cold pressing has also advantage of maintain or retaining more nutrients when compared to hot pressing using heat. This is the reason that heat pressed oil is less in cost when compared to cold press oil. But there is heat production in cold press due to friction produced in the expeller. But it must be seen that the temperature is less than the 70 degree Celsius to prevent the protein denaturation by heat.
Oil Extraction Unit
The Steps in Mustard Oil Production Process
- Drying of seeds:
The drying process is simple and takes place under the heat produced by the sunlight. Then for the storage purposes these dried mustard seeds are sent to silos. This step is mandatory and vital in the extraction of oil as there is a chance of oil deterioration in the presence of water.
Figure 1.0: After drying of the seeds the storage of mustard seeds in the silos.
- Inspection of the seeds and their cleaning:
There is a cleaning process for the stored dried seeds which were stored in silo are now transferred to Chalna for further cleaning activity. The seeds may contain dirt and foreign matter that may be effecting the yield of oil, so this dirt and foreign particles must be removed so that there is maximum yield without any impurities. That is why these are sent to Chalna to be made free from dirt and foreign particles. The cleaning process is devoid of any fluids or water. Buhler sorter is a seed sorter utilized for the cleaning of seeds.
Figure 2: For the cleaning and inspection purposes the instrument used.
- Seed crushing
For the extraction of oil as we know cold press or cold mills are used. This process involves the use of high power driven expellers and Ghanis. There are around twenty Ghani which are used each side which are power driven by two motors.
The seeds are fed to the Ghani which produces pungent oil. Also this method is an old mechanism for the extraction of oil. This Ghani comprises of mortar and pastel. The mustard seeds which were dried, cleaned and inspected are then fed to the kohlu. Due to crushing action the oil comes out. The kohlu moves in an circular motion. Due to this friction is produced between the kohlu’s body and the seeds. That is why oil starts to come out. The Ghani is at low speed so that temperature doesn’t exceed 50 degree Celsius. In cold pressing machinery or in the milling machinery crushing of the mustard seeds is done. In this cold press or cold mill process there is no use of extra heat or chemicals. It is natural and only cold press is used mechanically or hydraulically pressing or crushing the seeds to extract oil. For every 10 degree Celsius increase in temperature the chemical reaction becomes two times. The cold pressing has also advantage of maintain or retaining more nutrients when compared to hot pressing using heat. This is the reason that heat pressed oil is less in cost when compared to cold press oil. But there is heat production in cold press due to friction produced in the expeller. But it must be seen that the temperature is less than the 50 degree Celsius to prevent the protein denaturation by heat. To retain the pungency of mustard seeds into the oil, the maintained temperature must be less than 50 degree Celsius. It also retains the phytochemicals and micronutrients as the temperature doesn’t exceed 50 degree Celsius. Traditionally animals were used for the driving Ghani. But now its using electrical power to run.
The mustard seed when it is sent into kohlu and missed with water, there isa reaction which takes place. This reaction is that the thoglucoside which is a component of mustard seeds is enzymatically converted into allyl-iso-thiocyanate by the enzyme myrosinase. The AITC is very much vital component. It has anticarcinogenic activity and is a lachrymator and antbacterial also. The AITC is a lchrymator hence its effect is high, it is so much that the labour in there are not able to keep their eyes open. This process obtains or extracts about 23 percent to 26 percent of oil.
Figure 3: Ghani used in Puri oil mills limited.
Figure 4: Figure demonstrating the crushing of mustard seeds by the action of ghani.
- The expelling process:
About 23 percent to 26 percent of oil is extracted in the Ghani then the solid obtained is sent to the expeller and there is also a conveyer which is followed by a batch process. For a timeperiod of about an hour the seeds are in expeller which produces a large quantity of oil. This large quantity of oil is about 60 percent to 70 percent of oil approximately. The manual removal of the cake residue is done and then this is transferred or sent to the second expeller where the crushing of oil cake is done further. This process is done in a chamber with high pressure. Then in all the expellers the process is repeated. This is done to ensure maximum quantity of oil is extracted. The total yield of oil in the expelling process is in the range of 33 percent to 36 percent. This percentage or quantity depends on the seed quality such as moisture content.
Figure 5.0: Equipment used for extraction of oil in puri oil mills limited.
- Filtration process:
The final expelling process produces oil quantity in the range of 33 percent to 36 percent then the oil is stored in a tank called unfiltered tank. Here the oil is sent for the process of filtration using filter press. This process ensures that the remaining impurities present in the oil are eliminated from the oil.
Figure 6.0: Equipment used for oil extraction in puri oil mills limited
The filtration removes the last of the impurities present in the oil then the oil is sent for packing. This packing may be in the form of bottles or packets or tins. After this the bottles, tins or packets are printed with expiry and manufacturing date. This is also ensured then they are ready for sale.
Figure 7.0: Equipment used by puri oil mills limited for packaging and storing of oil.
6.0 MATERIALS AND METHODS:
There are various methods for the extraction of oil. Some are mechanical and some are hydraulic. But these methods are of high cost. Hence the use of biotechnology in combination with chemical methods are being researched to reduce the cost. These methods are being demonstrated in my project. The extraction methods used were
- Solvent Extraction
- Aqueous Extraction
- Enzymatic Extraction
- Quantity analysis of protein as a byproduct.
Experiment 1: Solvent Extraction Method.
- Hot Air oven
- Solvent extraction using soxhlet apparatus.
- Electric balance
This is a selective procedure for extraction of oil and protein is also obtained from the deoiled cake. In this process the desired compound is mixed with an solvent or organic solvent containing a reagent that is vital for extraction. Here the substance which is of interest reacts with the solvent and it is more soluble in organic solvent than in aqueous solution. Then a recovery process is followed to recover the product.
1. Crushed the seed sample in mixer.
2. Transferred and weighed 20g material to an extraction thimble, avoiding any loss.
3. Placed the thimble in an extractor previously fitted.
4. Added sufficient quantity of solvent i.e. petroleum ether and affixed the condenser and allowed through it a cold water current to be flowing and heated the extractor so that the action was moderate not violent.
4. Continued the extraction for 8 hours. Then removed the extraction from it’s bath, taken the extraction thimble, after it has been drained or extracted out of the extractor & is allowed or kept to evaporate from it in a current of air.
5. Recovered the solvent. Use distillation to remove the greater part of solvent..The flask is heated in the hot air oven to remove the organic solvent for about a timeperiod of one hour and at a temperature 105 degree Celsius. After cooling the flask to room temperature, the weight is recorded.
This process of heating is repeated till the weight of the flask doesn’t fluctuate or becomes constant.
Oil being extracted.
Figure 21: Solvent Extraction apparatus.
Figure 22: Crushed Mustard seed. Figure 23: Extracted oil.
Figure 24: Extracted oil dried in desiccator.
Experimental Design of Solvent Extraction Process
|Extraction time (h)||A||8||8||8|
|Mass of sample (g)||B||20||20||20|
|Solvent volume (ml)||C||250||160||180|
|Experiment number||Sample weight in gm||Weight of thimble.
|Weight of flask without oil in grams. ( Wo)||Volume of petroleum ether in ml||Weight of flask with extracted oil in grams. (Ws)||Weight of oil extracted.
|Percentage of oil extracted.(%)|
Table 3.0.0: Percentage of oil obtained by solvent extraction method.
Results and discussions:
Graph 1.0.0: Graph between experiment trial used in solvent extraction and percentage of oil extracted.