ABSTRACT This study aims to develop an efficient technology in extracting essential oil from lemongrass (Cymbopogon citrates) that

will increase the oil recovery, improve oil quality and stability. The essential oil was extracted using the enzyme pectinase. A control and single treatment were manipulated. The freshly harvested lemongrass were processed, then the enzyme (pectinase = 0.5% of the sample+ buffer ) was added to the treatment while only the buffer was added to control (1:6 sample: buffer dilution ratio). After 12 hours of incubation at room temperature with constant shaking, the reaction mixtures were boiled in a water bath to inactivate the enzyme. When cooled, the mixtures were filtered in cheesecloth and extracted with a solvent. The collected organic layer was subjected to the rotary evaporator or electromantel oil extraction evaporator to recover the essential oil. The oil was then filtered busing filter paper. An essential oil yield of 0.96% f the raw sample was obtained for the treatment and 0.47% oil yield for the control. Prior to the physico-chemical analysis, the enzyme-treated lemongrass oil can be considered similar to the of an extra grade oil having a specific gravity of 1.033), solubility in 70% ethanol of 10:1 ratio and a strong lemon-like odor as perceived by the panelists. The untreated sample was of low quality oil (based on the related literature). Given the results, it may be concluded that essential oil production through pectinase application could be answer to the countrys high dependenc on imported essence.

CHAPTER 1 THE PROBLEM AND ITS BACKGROUND Introduction Essential oils are very much in demand in the Philippines, especially in the pharmaceutical and cosmetic industries. These industries heavily depend on foreign essences and cosmetic preparation for equate supply and are spending millions of dollars for the import of essential oil and other toilet Preparation (Manalo et al.,1982). The Philippines rank 8th among the importing countries, garnering 1.3% of the worlds share and practically Imports more than 90% of the countries requirements (Dar, 1997).

High dependence of the Philippines on imported essential oil increased From US $ 37.2 million in 1989 to US$ 59.3 million in 1993 while exports Decreased from a value of US$ 2.4 million in 1989 to US$ 1.5 in 1993; in terms of quality (net kg) from 328,494 kg in 1989 to 180,084 kg in 1993 (Jamilla, 1997). Data for the period 1989-1993 show an increasing trend value of importation of essential oils (De Guzman, 1997). Given the outlook for the continuing economic growth of the country, one will assume that the demand for essential oils is also likely to grow, as more money become available for discretionary funding. It is therefore timely and relevant to further develop and explore the essential oil bearing plants to lessen the countys dependence on imported essential oils (Jamilla, 1997). Moreover, lemongrass is a very important oil component in soaps, bath salts, sprays and perfume preparation. It is also use for the isolation of citral which is employed in artificial flavors and in the manufacture of ionones. In addition, lemongrass oil is also used as starting materials for Vitamin A. Lemongrass oil is obtained by methods such as steam distillation, Extraction by volatile solvent, expression by hand or machine and enfleurage. Steam distillation and solvent extraction are used commercially. However, the processes produce only small amounts of oil, thus, have a low cost effectivity. On the other hand, the use of pectic enzyme in various industries is increasing. It is enery-efficient and economically feasible (Espino, 1997). This study therefore was conducted in order to develop a new technique, the pectinase application, in extracting essential oils from lemongrass so as to increase its yield, quality and stability. The Research Objectives This study focuses on the production of essential oils from lemongrass through enzyme process. Specifically, it aims: 1. To extract essential oils from lemongrass through enzymatic process under optimized condition; 2. To conduct physico-chemical analysis of the extracted essential oils; 3. To compare the essential oils yield between the control and enzymetreated samples; and 4. To compare the physico-chemical properties of the control and enzymetreated samples.

Theoretical/Conceptual Framework INDEPENDENT VARIABLES Control (without enzyme) Treatment 1 (w/ enzyme) DEPENDENT VARIABLES The yield, quality And stability of Lemongrass oil

Fig. 1.1 Paradigm of the Independent and Dependent Variables of the Study Pectinase-enhanced Production of Essential Oil from Lemongrass (Cymbopogon citratus) Scope and Limitations of the Study The study was conducted at the Immunology and Enzyme Laboratory of the National Institute of Molecular Biology and Biotechnology of the University of the Philippines, Los Baos, Laguna from May 1-31, 1999. The second trial was conducted at Cincos Residence and the Filipinas Palm oil Plantations, Inc. Laboratory from August 28- December 29, 1999. The study focuses only on the effect of the enzyme pectinase on the production of essential oil from lemongrass. The researcher was concerned in determining the percent essential oil yield and in performing its physic-chemical analysis. The researchers also utilized the available laboratory apparatus such as the Electromantel oil extraction evaporator equipment of the FPPI palm oil mill laboratory. Data gathering and statistical analysis has the limitation of bias. Significance of the Study The Philippines is dependent on importance of essential oils from Europe and the United States, despite the fact it is one of the tropical countries rich in essential oil-bearing plants (Anzaldo, 1982). Our country practically imports more than 90% of our requirements and ranks 8th among the essential oil importing countries (Dar, 1997). The traditional method of essential oil extraction is by steam distillation. However, the process involves the use of much energy and it yields small quality of oil apart from loosing some of its components. This study was conducted to develop a new technology in extracting essential oils from locally available lemongrass that will account for maximum yield, better quality and stability; thus, alleviating the supply of essence and coping with the increasing demand. This project can be considered as an initial phase to scale up the production of essential oils using the pectic enzymes previously established by the BIOTECH. It can be the basis for other pectinase applications.

The use of pectinase has several advantages in contributing to a more economical process. As it is economically feasible, a large scale production for commercial purposes will soon be possible in the country. This is also in support to the long standing concerns of economic stagnation in the rural areas of many developing countries by the United Nations. The program for the development of the essential oil industry was perceived to answer these concerns. As stressed by UNIDO, it is an agro-based industry that can utilize rural sector participation in the cultivation and harvesting of raw materials and to some extent, even in field of distillation activities. The improvement of the agro-techniques and the development of energyefficient and affordable technology in the extraction process can add value to the product and increase its market potential. It can also be a strategy in solving the importation problems of foreign made essential oils. Definition of Key Terms Aldehyde - production by the oxidation of alcohol compound containing the CHO group and used in the manufacture of dye and synthetic rubber. Buffer - a substance capable of maintaining the relative concentrations of hydrogen and hydroxyl ions in a solution by neutralizing; capable of maintaining the pH of a solution. - an unsaturated liquid aldehyde C9H15CHO that has a strong lemon and verbena odor; used in flavoring and in perfumery and consists of a mixture of two stereoisometric forms. - any of a very large class of complex proteinaceous substances that are produced by living cells that are essential to life by acting like catalyst in promoting chemical reactions in the cell without being used up; useful in many industrial processes. - combination of organic acids and alcohol; sweet smelling compounds widely used as artificial flavors and dye. - product of the reaction of two molecules of alcohol; very volatile and highly flammable. - any of five isometric volatile liquid paraffin hydrocarbons found in petroleum especially the normal hydrocarbon.

Citral

Enzyme

Esters

Ethers Hexane

CHAPTER 2 REVIEW OF RELATED LITERATURE The Pectic Enzyme and Aspergillus Family Pectinases are produced by molds, yeasts and bacteria. Molds especially the species belonging to the Aspergillus family, are used for commercial production of these enzymes. Espino et al. (1989) partially isolated pectic enzyme extracellularly-produced by Aspergillus ficuum and A. niger isolated from pomelo (Citrus grandis Osbeck). The enzyme produced high pectolytic and clarifying activities. Espino et al. also studied the pectic enzymes of A. ficuum isolated from local agricultural wastes. Several studies have been done describing the purification and properties of pectic enzymes produced by Aspergillus sp. Isolated from various substrates. Pectic substances are found in the wall, intercellular spaces, juices and saps of higher plants (Whitaker, 1972). They can hamper processing or lower the quality of a product, hence, pectic enzymes causes these substances to easily degrade. The enzyme (pectinase) used in the study was provided by the group of Dr. T.M. Espino at BIOTECH UPLB. Pectinase was isolated from Aspergillus niger grown by solid substrate cultivation in enameled trays placed in cabinet type fermentor. Economic analysis of pectinase production showed that, the cost per ml and cost per pectin transeliminase (PTE) unit of crude pectinase was PO. 087 and P 0.034 respectively (Espino, 1997). Studies on Pectinase Application Pectic enzymes, which degrade pectic substances, are of major importance to food industry. They are utilized in the fruit juice industry to facilitate extraction and clarification of juice (Forgat and Kelly, 1983; Whitaker, 1984). Pectic enzymes are also used for maceration and liquidation of fruits and vegetables. Today, using pectic enzymes, it is possible to extract juice fruits from fruits such as banana, guava, or papaya without addition of too much water that affects the characteristics of fruit flavor and aroma. Pectic enzymes ca also be utilized in the pre-treatment of pectic wastewater. (Tanabe et al., 1986). Another important industrial application of pectinases is in the degumming of fibers crop like ramie (Deshpande and Gurrucharanan, 1985). During stepwise extraction of pineapple juice, enzyme addition also improves the quality of juice by allowing extraction of more soluble solids and juice particles. In the industrial processing of plant tissues, these enzymes have been adopted to improve the yield of alginate base materials from harvested seaweeds, remove mucilage in coffee after fermentation, pretreat cereals, beans and pulses to reduce cooking times and temperatures, accelerates rotting of flax, macerate vegetables and other applications (Godfrey, 1983).

To increase the oil yield and improve its quality, Montedro et al. (1993) investigated on the pectinase from olive oil vegetation waters and its use in the mechanical olive oil extraction process. An increase of 8.9% in olive oil yield and generally improved quality, turbidity, oxidation, induction time, chlorophyll and contents of aromatic compounds resulted from the study. The UPLB BIOTECH produced pectinase was also used for the extraction of essential oils from ilang-ilang flowers. Results of the experiment showed that there was an increase of about 75% in the yield of the enzymetreated sample and the quality was considered similar to that of the extra grade oil while the untreated sample are of lower quality essential oils. (Espino et al., 1997). The Essential Oils Essential oils are volatile constituents of plant material, which collectively are responsible fro imparting the characteristics odor associated with the plant material itself (Wijieskera and Tatnatunga, 1993). Their almost complete volatility in steam distinguishes them from so called fixed or fatty oils such as coconut oil or palm oil, which are derivatives of glyceric acid, the essential oils are mixtures of a variety of chemical ranging in number from few to several. They are also sometimes called volatile or essential oils. Essential oils vary from colorless to yellow or brown and are usually a mixture of compounds. The compounds occurring in essential oils are as follows: (1) hydrocarbon, (2) alcohol, (3) esters, (4) aldehyde, (5) ketone, (6) lactones, 97) phenol, (8) acids and (9) terpenes. The importance of essential oils can be gleaned from their extensive utilization in various industries. They serve as major and minor constituents in the manufacture of perfume and cosmetics, flavoring agents in food and confections and pharmaceuticals for treatment of ailments. Current Market of Essentials Oils. Verlet (1993) estimated that world production of essential oils is around 45,000 tons, valued at US$ 700 M. The Philippines is one of the principal countries that is very dependent on imports from China, United States of America, Brazil, Indonesia, India and Europe. In 1989-1993 there is an increasing trend in the value of importation of essential oils. The highest value and amount of export was observed n 1989. Our export of essential oils declined thereafter but rose again in 1993. Based on the data, we have been exporting oils of peppermint, spearmint, lemon, etc. but these commodities are most likely re-export materials as we do not have any local, commercial production of these oils. (Based on the statistical data obtained from the Foreign Trade Statistics, Department of Trade and Industry). All these figures combined show our tremendous dependence on importation of our essential oil needs. Our net import of these commodities amounted to US$ 58 M for 1993 (Jamilla, 1997). Import substitution in the traditional sense of replacing imported essential oil with domestic sources of production and supply, accompanied by the building up of tariffs and quotas on importation, is likely to be considered by our policy-

makers to be out of tune with the trade globalization and liberation measures that have been adopted by the government (Jamilla, 1997). The low priority that has been given to the development of essential oils industry is what makes the development more challenging. This is the real challenge that undoubtedly calls for a concerted multidisciplinary approach. As the Dean of the College of Agriculture said there was money in the essential oils business for even hunger was no excuse for a person not to smell good. Extraction of Essentials Oils. Essential oils are usually recovered from plants by two methods, either by distillation or by solvent extraction. Solvent extraction is technically a more advanced process and yields real representative odors, but is more expensive than distillation. Steam distillation, on the other hand, is a very much more gentle process and causes few drastic changes in the oil. During the process, the collected mixture may consist largely of steam with only small portion oil. Distilled oils may also be considered artifacts, as they may or may not be the same components, which are lost during the process. It is therefore advisable to engage in new technologies and techniques in the extraction of essential oils that would be cheaper and increase the oils yield and stability. The Lemongrass Oil Literature about the nature of essential oil found in lemongrass is very limited due to lack of knowledge that it can be a substitute source of essential oil. Lemongrass, locally known as tanglad, contains oil having a strong, lemon odor due to its citral content. It is widely used as flavorant for food and pharmaceutical industries. It is not only known for its essential oil but also for the medicinal value of the palnt itself (Coronel et al., 1984). The physio-chemical properties of lemongrass oil based on the study conducted by Torres (1994) are as follows: (1) refractive index at 20oC = 1.4892; (2) specific gravity at 20oC = 0.8876 to 0.9215; (3) solubility in ethanol (v/v in 70%)=1:1; (4) acid value = 11.04 to 14.14 mg/g: and citral content by GC = 75% minimum. The oil is useful in flatulent and spasmodic affections of bowels and in gastric irritability. It is effective as a liniment for chronic rheumatism, limbago, nueralga, sprains and other painful affections and ringworm (Quisumbing, 1978). It has hypotensive property which lowers blood pressure when used in the experimental animals. Lemongrass oil is a main source of citral for lemony and verbena notes, as well as for chemical transformation into ionone, mythyl ionone, etc. the oil contains at least 75% citral (Bellanato and Hidalgo, 1984). Ionones are now employed for the manufacture of synthetic Vitamin A. Aldehyde content contributes to the aging of essential oils.

CHAPTER 3 METHODOLY The Research Design The experimental design used in this study was a two-group design wherein a control and a single treatment were manipulated (Table 3.1) there were eight trials conducted both in the control and the treatment. Table 3.1 Research Design Composed of the Control and the Treatment CONDITIONS Lemongrass Enzyme (pectinase) CONTROL 500 g absent TREATMENT 500g 2.5 g (61.60 PTE units 0.5% of the sample 12 hours 28oC 1:6

Incubation Time Incubation Temperature Sample: buffer dilution Ratio Source of Materials

12 hours 28oC 1:6

Freshly harvested lemongrass were obtained from Los Baos and Quezon (for trials 1, 2 and 3) and Purok 3 Brgy. 3, San Francisco, Agusan del Sur (for trials 4 to 8). The pectic enzyme used in this study was provided by the group of Dr. Espino of the National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baos College, Laguna. The average pectinase transelinmase activity of the enzyme used in this study was 24.64 units per gram of ammonium sulfate precipitated pectinase. Enzyme Treatment Freshly harvested lemongrasses were processed immediately for extraction. The lemongrasses were cut into pieces and ground in an osterizer.

The samples were weighed and then the enzyme solution was added. The enzyme solution was prepared by dissolving the enzyme in 0.2 M acetate buffer, pH 4.0 at !:6 sample: buffer dilution ratio. The reaction mixture was incubated at 28oC with constant shaking for 12 hours. A control consisting of the ground lemongrass leaves and buffer was also prepared. The reaction flask was sealed tightly to prevent any loss of essential oils. After incubation, the reaction was stopped by placing the mixture in a boiling waterbath for 10 minutes to inactive the enzyme. Lastly, the reaction mixture was cooled to room temperature before extraction. Extraction Procedure The reaction mixture composed of the sample, enzyme solution and solvent were filtered using cheesecloth. Manual squeezing was done to recover most of the liquid. However, for maximum market production of lemongrass oil, the use of machine operation is highly recommended. The filtrate was collected while the residues were discarded. The extract was transferred into a weighed flask. The flask was connected to the rotary evaporator system to separate/recover the solvent from the essential oil. The process was stopped when the content of the sample flask was perceived viscous (for Trials 1, 2, & 3). For trials 4 to 8 the solvent was recovered from the essential oil by the use of an Eloctromantel oil extraction evaporator equipment in the oil mill laboratory of Filipinas Palm oil Plantations, Inc. The extracted essential oil is then filtered using filter paper. Data Gathering Technique After the extraction process the essential oil recovered from lemongrass was weighed. The percent yield of the oil was the determined using the formula: Percent essential oil yield = weight of essential oil x 100 weight of the plant sample The process was used from the second to the eight trials both in the control and enzyme-treated samples. In measuring the percent yield of the lemongrass oil, the researcher used the available analytical balance. The researcher also subjected the sample to physic-chemical analysis. The results were compared to the literature value as the standard basis for comparison. Twenty panelists were invited to evaluate the odor rate of the oil. The panelists were chosen for their knowledge on essences. Samples for each trial, both in the control and treatment, were placed in a single room for the panelists to evaluate. The panelists were given score sheets for rating the odor using the 5-point rating scale.

Data Gathering Method The results were gathered and tabulated. Quantitative and qualitative analysis were done based on the following: 1. Comparison of essential oil yields between the untreated and enzymetreated samples. 2. Quality analysis of the essential oil. The extracted essential oil from lemongrass was analyzed as to its specific gravity and solubility. The values obtained were compared with those reported in the literature. Arithmetic mean, t-test and analysis of variance (ANNOVA) were the statistical tools used in analyzing and interpreting the data. Mean was used to determine the average of the percent yield of the oil as well as its rate. ANNOVA and t-test were used to determine the significant difference of the results.

FLOW CHART OF THE PROCEDURE Enzyme preparation (enzyme (2.50g) 0.5% of the sample + 3L, 0.2 M buffer. pH 4.0) Processing of Lemongrass (cutting into pieces and grinding using an osterizer) Addition of enzyme solution (1:6 sample: buffer dilution ratio) Incubation of reaction mixture at room temperature For 12 hours with constant shaking Boiling in waterbah for 10 mins. to inactive the enzyme Cooling to room temperature Filtering with cheesecloth Extracting with petroleum ether as solvent Recovery of organic layer in a separatory funnel Recovery of essential oil by rotary evaporation or Electromantel oil extraction evaporator Filtering with filter paper Weighing of recovered oil and determining the percent yield Physical and chemical analyses of the recovered essential oil

Chapter 4 RESULTS, ANALYSIS AND INTERPRETATION OF DATA The results of this study were shown in the preceding tables and figures. Comparison of Essential Oil Yields between the Control and Enzyme treated Samples Previously established optimum conditions were adopted in extracting essential oil from 500g lemongrass (Galang, 1997). Results showed an essential oil percent yield of 0.47% for the control and 0.96% for the enzyme treated samples (Table 4.1) were obtained. The oil yield of the enzyme-treated sample was approximately twice than that of the control (without enzyme). Moreover, the oil yield obtained in this study for the enzyme-treated samples (0.96%) exceeded the literature range value of 0.48% to 0.78% of the total weight of the sample. Yield of 0.2% to 0.4% can be considered normal (Brandares, 1987). There is a significant difference on the average percent essential oil yield between the control and the enzyme-treated (2.987**). the computed t-value exceeded the tabular F-value in the 5% and 1% probability. Based on the results obtained, application of pectinase on essential oil extraction truly enhances and increases the essential oil yield. The use on enzymatic process could be the key to provide the growing demand of essential oil in the industry today. Furthermore, with the use of a minimum amount of enzyme (0.5% of the sample, 61.60 PTE units) at a sample to buffer dilution ratio of 1:6 (w/v), under 280C and for 12 hours incubation time, essential oil extraction from lemongrass is feasible. Large-scale extraction of essential oil from lemongrass could be explored and feasibility studies on economic viability of the process in order to attract investors. Physico-chemical Analysis of the Essential Oil from Lemongrass The extracted essential oil was a golden yellow colored liquid (deepest color base on the color range indicated in the literature) and has a lemon- like odor, the characteristic fragrance of lemongrass. Based on the physical appearance, the enzyme treated oil appeared to be darker than the oil obtained from the control sample. In addition to these, other physical and chemical properties were also examined and compared. Table 2 presents the physicochemical properties of the extracted essential oil lemongrass. The specific gravity is directly related to the quality of the oil. Results of the analyses showed that the enzyme treated lemongrass oil had higher specific gravity (1.033) than the control (without enzyme) with only 0.676. The use of pectinase showed that enzyme treatment5 improved the quality of lemongrass oil. Based on the literature value (Robbin, 1983), the specific gravity of the enzyme- treated lemongrass belongs to the extra grade oil category. However,

the value is a little bit lower compared to the commercial oil. On the other hand, the solubility in 70% ethanol (v/v) of the enzyme-treated lemongrass oil is the same as that of the literature value. As perceived by the 20 panelist, the enzyme-treated lemongrass oil has a higher odor rate compared to that of the control. Based on the results of the physical and chemical analyses, enzyme treatment resulted to better quality essential oil as compared to the control. At the same time, the increase in essential oil yield as a result of enzyme treatment compensated the cost of the enzyme added. Therefore, it can be concluded that pectic enzyme treatment under optimized conditions at laboratory scale can be an alternative method for essential oil extraction of lemongrass on large scale. Table 4.1 Comparison of the Percentage Essential Oil Yield of the Control and Enzyme-Treated Lemongrass Trial 1 0.64 0.98 Trial 2 0.41 0.94 PERCENT YIELD Trial Trial Trial Trial 3 4 5 6 0.39 0.95 0.63 0.96 0.23 0.99 0.42 0.94 Trial 7 0.63 0.98 Trial 8 0.38 0.96

Treatmen t Control Enzymetreated

Average 0.47 0.96

Conditions used:

0.5% enzyme concentration 12 hours incubation time 280C incubation temperature 1.6 sample: buffer dilution ratio : : 2.987** t 0.05(8) = 1.860 t 0.25(8) = 2.0306 t 0.1(8) = 2.896

T-test values: Computed t-value Tabular t-value

Chapter 5 SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS Summary of Findings Essential oil from lemongrass was extracted using the pectinase under the following optimized conditions: enzyme concentration: 0.5%, incubation time: 12 hours, incubation temperature: 280C, sample: buffer ratio 1:6 (w/v). An essential oil yield of 0.96% was obtained for the enzyme-treated sample while 0.47% oil yield for the control (without enzyme). This shows that pectinase application doubles the yield of the extracted essential oil from the lemongrass. Moreover, there is a significant difference on the mean of the control and of the enzyme-treated samples. Results on the physical and the chemical analyses of the extracted essential oil showed that enzyme treatment improved the quality of the oil compared to the control (without enzyme). The pysico-chemical properties of the enzyme-treated lemongrass oil were as follows: specific gravity = 1.033 and solubility in 70% ethanol = 1.1 (v/v). these properties were comparable with those reported in the literature. On the other hand, there is also a significant difference on the odor rate of the lemongrass oil as perceived by the 20 panelists. Conclusion The application of the pectinase in the extraction of essential oil from lemongrass was found effective. Therefore, the researcher believed that the enzyme treatment increased the essential oil yield and further improved its quality and stability. Moreover, the use of enzymatic treatment contributes to a more economical process and is therefore economically viable. Thus, the application of peptic enzyme at laboratory scale could be an alternative method for essential oil extraction on a large scale.

Recommendations Production of essential oil from lemongrass through enzymatic process yielded positive results. With this basis , the researcher recommends the adaptation of this new technology on the extraction of essential oils. To further improve the study, the researcher also recommends the following: 1.Characterization of lemongrass essential oil components should be made in order to determine which is responsible for the characteristics of lemongrass odor; 2.Other physical and chemical analyses should be conducted on the extracted essential oil to further evaluate its quality; and 3.Large-scale extraction of oil from lemongrass through enzymatic process should be further explored and feasibility studies on the economic viability of the process should be conducted. Furthermore, application studies on other oil-bearing plants apart from lemongrass should be explored.

References Adegoke, G.O. and B.A. Odesola, 1994. Storage of maize and cowpea and inhibition of microbial agents of biodeterioration and biodegradation. 6(2): pp. 81-84. Bellenato, J. and Hidalgo. Infrared Analysis of Essentials Oils. Heyden and Son Ltd. Madrid, p. 1035 Brandares, M.F.T. A.M. Vuelban, B.B. Darjuan, M.R. Ricalde and F.E, Anzaldo. 1987. Stability Studies of Essential oils from Philippine Plants. II Cymbopogon citrates (DC) Stapf. Phil. Jou. Sci 116 (4):391-402. Coronel, V.Q., F.E. Anzaldo and M.P. Recaa, 1984. Effect of moisture content on the essential oil yield of lemongrass and citronella. NSTA Technology Journal IX (3):36-28. De Guzman, C. Horticulture Production of Essential Oil Plants in the Philippines Paper presented in the 1st Southeast Asian regional Conference and Exhibition on Natural and Herbal Products (SARCRNHP) in June 1997. D. Guzman, C. Annual Report on Essential Oil Research and Development Program (DOST-PCCARD). July 1993- June 1994. Espino, T.M., P.C. Sanchez, et al. Pilot Production and Utilization of Peptic Enzymes from Food Applications. Annual Report. June 1995- Nov. 1997. Forgarty, W.M. and C.T. Kelly, 1990. Microbial Enzyme and Biotechnology. 2nd ed. Elsevier Science Publishers, Ltd. England, pp. 290-291. Galang, M.C. 1999. Application of Pectinase on the Extraction of Essentials Oils from Lemongrass (Cymbopogon Citratus). Jamilla, B. Prospects of Essential Oil Business in the Philippines. Paper presented to the 1st SARCENHP in June 1997. Manalo, J.B., V.Q. Coronel and F.E. Anzaldo. 1982. A new development in cologne formulation using ilang-ilang flower oil. J. NSTA Technology. 7 (2): 21-34. Montedro, G.F., A.L. Begliomini, et al 1993. Pectinase production from olive vegetation waters and its use in the mechanical extraction to process increase oil yield and improve quality. Italian Journ. of food Science 5 (4):355-362.