Enzymatic properties of microbial solid starters on coconut oil recovery

ISBN 978-979-18962-0-7 Enzymatic properties of microbial solid starters on coconut oil recovery Rita Dwi Rahayu, Joko Sulistyo, Achmad Dinoto Bidang Mikrobiologi, Pusat Penelitian Biologi, Lembaga Ilmu Pengetahuan Indonesia LIPI Jl. Raya Jakarta-Bogor Km 46, Cibinong Science Center, Cibinong 16911 Indonesia Phone: +62-21-8762066; Fax: +62-21- 8765062 Abstract To understand the enzymatic capacities for coconut oil extraction, three microbial isolates representing Aspergillus oryzae K1A (mold), Candida rugosa K2A (yeast), and Lactobacillus plantarum K3A (bacteria) were compared. We confirmed that all tested strains produced amylase, protease, and lypase. However, among the tested strains, K3A shows the highest activities of amylase, protease and lipase reaching 1.48 IU/ ml, 2.24 IU/mL and 2.01 Iu/ml, respectively, under the laboratorial condition. As predicted, those enzymes are related to oil extraction yielding the highest oil recovery from coconut milk as much as 23.5% (v/v). Keywords: Coconut oil, enzymatic extraction, Aspergilus oryzae,candida rugosa, Lactobacillus plantarum, solid starter Introduction Technical development on improving coconut oil quality is increasing and one of them is by application of enzymatic fermentation where is the product is then reffered as Virgin coconut oil (VCO) (Rosental et al. 1996; Rindengan & Novarianto, 2004). VCO is a coconut oil that is produced by such this kind of process. It contains high lauric acid (approximately 50%) belongs to saturated fatty acid with a medium chain (C-12) and commonly known as medium chain fatty acid (MCFA). Coconut oil that was produced by enzymatic process has a property of low free fatty acid and low water content (0.02 0.03 %), clear, good smell and lifetime might be more than one year (Rindengan & Novarianto, 2004). The production of VCO by enzymatic fermentation includes the usage of yeast, microbe or the other enzymes those are effective to break down coconut emulsion. The activity of enzyme is influenced by substrate and enzyme concentration, temperature, ph, and incubation time for enzymatic reaction (Palezar & Chan, 1986). Microbial strain was a major choice to accelerate the process since it performed proteolytic, amylolytic and lipolytic activities. These enzymes were needed to hydrolyze protein, carbohydrate and fat components to stabilize coconut emulsion. The basic principal of enzymatically coconut oil production is to make the emulsion becoming unstable and the content of carbohydrate and protein are therefore coagulated and released oil from the emulsion naturally with a very little change occurred on its chemical composition (Sulistyo et al, 1999). The purpose of research is to examine the capacity of microbial solid starters on virgin coconut oil recovery through enzymatic fermentation process. Materials and Methods The research was conducted on two steps were production of solid starter using the three respecting isoates were Aspergillus oryzae (K1A), Candida rugosa (K2A) and Lactobacillus plantarum (K3A) and application of these starters for production of VCO. Production of solid starter Solid starter was produced by using three isolates were K1A, K2A and K3A and incubated at room temperature for 4 days on 100 gram steamed rice that had been presubmerged for 24 hours and mixed with 1% wood ash. After completely incubation by the procedure the starters were dried at 40 o C for 3 days and than grinded and observed for colour, smell, texture and their enzymatic activities. Production of VCO Coconut milk was prepared by extracting a bowl of shreeded coconut with hot water (3:1,w/v) and the obtaining coconut cream was than inoculated with 1 % solid starter after separating from coconut skim one and incubated at 43 C for 10-15 hours. There were three phases formed after overnight incubation those were oil, protein and water phase, where the coconut oil was on the top of layers. After separating and screaning, coconut oil was then observed for yield, 648

colour, smell, and then analyzed for ß-karoten, triglyserida, and organic acids content. Procedure for organic acids analysis (Bevilacqua & Caifano 1989) Approximately 5 g sample was added to 50 ml acetonitril buffer. The buffer was prepared by mixing 0.4 % acetonitril solution with 0,5% (NH 4 ) 2 PO 4 solution into a water containing H 3 PO 4 until ph was 2,24. The sample was mixed and homogenized with the buffer for an hour continuously, after centrifuging at 7000 rpm for 5 min, supernatant was then filtered twice through a filter paper and continued filtering through a filter membrane with porous size of 0.45 µ just before injecting to HPLC. Yield of Virgin Coconut Oil The yield was determined by gravimetric method as follows: Yield = Volume of obtaining oil (ml) x 100% Volume of coconut cream (ml) Moisture content Ten gram of VCO sample in triplo were respectively placed onto petri dishes and weighed to measure their initial weight and then removed onto a drying oven at 105 C for 2 hours and immediately remove onto a desicator for approximately 15 min and weighed again to determine each final weight. (Suminar et al, 2001) Moisture content = A B x 100% A A = initial weght of sample before drying B = final weght of sample after drying Preparing for crude amylase Ten gram sample of each treatments were added with 20 ml 0,1 M Mac Ilvane buffer at ph 7,0 and homogenized. The homogenats were then centrifuged at 9000 rpm for 10 min and their filtrates were used as crude amylase. Analysis of amylase activity Amylase activity was measured by DNS method using solube starch as its substrate and maltose solution was used as its standard. Sample was prepared by addition of 0.1 ml crude enzyme with 0.9 ml BFS and immediately added with 1,0 ml soluble starch and mixed well and then incubated using a shaker at 50 rpm, 50 C for 30 min. After addition with 3 ml DNS reagent, the reaction was stopped by boiling for 5 minutes under waterbath condition and cooling down to room temperature. It was then added with 9.0 ml doubled distilled water and mixed well to obtain a gradation of colour and absorbance was measured at λ 550 nm using spectrophotometer. Blank or control was prepared using 1,0 ml soluble starch and mixed well and then incubated at 50 C for 30 min on the shaker at 50 rpm. After reaction with 3 ml DNS reagent, it was added with 0.1 ml enzyme solution and 0.9 ml BFS and stopped by boiling for 5 min. After cooling down to a room temperature, it was added with 9.0 ml doubled distilled water and the absorbance was read at λ 550 nm. One unit of enzyme is equal to the activity needed for releasing 1 µmol maltose per minutes per ml enzyme. Preparing for crude protease Five gram sample of each treatments were added with 10 ml of 65 mm phosphate buffer at ph 7,0 and homogenized. The homogenats were then centrifuged at 9000 rpm for 10 min and their filtrates were used as crude protease. Analysis of protease activity Protease activity was measured by using casein as its substrate and L-tyrosin 1,1 mm was used as its standard. Sample of enzyme solution (1,0 ml) was added with 5 ml reagent B into a tube. The reaction mixture was then incubated on a shaker at 37 C for 30 min, 200 rpm. Approximately 6 ml of reaction mixture was sampled and then centrifuged at 100.000 rpm, 4 C for 5 min. Blank sample was prepared as the procedure by addition of 5 ml reagent C with 1,0 ml enzyme solution and incubated on the shaker at 37 C for 30 min, 200 rpm. Approximately 6 ml of reaction mixture was sampled and then centrifuged at 100.000 rpm, 4 C for 5 min. To rise a colour development 2 ml of each samples were place on two separate tubes and then added with 5 ml reagent E and 1 ml reagent D for each. Both of solutions were then incubated on the shaker at 37 C for 30 min, 200 rpm. The absorbance of both solutions were measured at λ 660 nm using spectrophotometer. Results and Discussion Observation on properties of solid starters of VCO those were produced by the procedure as mentioned above was shown in Table 1 and Figure 1. It was found that among of the solid starters, the VCO produced by using the starter of K3A showed higher yield than that of the other two starters were K2A and K1A, where the yield by using the K1A starter showed the lowest. We have found from organoleptic test that had been done, that all of the tested solid starters showed a similar colour to each other, eventhough, the starter of isolate K3A (Lactobacilus plantarum) showed has the finest texture among of three, while K1A (Aspergillus oryzae) showed has coarse texture since it grown on a 649

solid substrate where its micellia spreadly penetrated to coagulate all particles of the substrate as shown on Figure 1. To know wether there is an influence of cell density or not on the yield of VCO, we have finally found an optimal concentration for solid starter to produce high yield VCO by influencing it with 1.0% concentration of starter. Table 2 exhibited the yield of VCO were produced by respecting microbial starters those were mentioned above. The highest yield was obtained by using K3A starter derived from Lactobacillus plantarum (23.5%), since it showed the highest also in producing amylase and lipase, followed by K1A starter (21.5%) and K2A starter (21.0%). The present of these enzymes available in the starters were directly effected to break down the coconut emulsion. The lowest yield was come from control (15.3%) that was no microbial starter involved in the process. It was found from visually observation on all VCO products those were extracted by using these starters indicated a fine and natural appearance from all their solid and liquid waste products. Those were light for solid waste and milky for liquid waste. Tabel 1 Properties of solid starters of VCO Solid Starter Enzyme (IU/ml) Colour Texture Amylase Protease Lypase K1A 1.23 1.73 1.5 Yellowish Coarse granule K2A 1.19 2.02 1.77 Yellowish Small granule K3A 1.48 2.24 2.01 Yellowish Fine granule K1A : Solid starter using Lactobacillus plantarum K2A : Solid starter using Candida rugosa K3A : Solid starter using Aspergillus oryzae Figure 1 Product of VCO starters using isolate of K1A, K2A, and K3A (from left to right) Table 2 Organoleptic test of VCO using different starters Solid Starter Concentration (%) VCO K1A 1 White K2A 1 White K3A 1 White Control 0 White Product and Waste Solid waste Liquid waste Flavouring VCO Yield (%)(v/v) Slightly oil flavour 21.5 Slightly oil flavour 21.0 Slightly mild 23.5 Slightly oil flavour 15.3 650

Tabel 3 Analysis of VCO using solid starters of isolates K1A, K2A, and K3A Fatty Acid Product of VCO (%) K1A K2A K3A Control Acetic 19.0 21.0 24.0 9.0 Lactic 79.0 80.0 81.0 16.0 Pyruvate 21.0 46.0 49.0 36.0 Malic 14.0 18.0 19.0 21.0 Oxalic 13.0 14.0 12.0 9.0 Caproic - 0.71 0.33 - Caprylic 5.53 7.90 7.91 9.30 Capric 6.53 2.68 2.89 8.03 Lauric 41.59 47.86 48.76 40.96 Myristic 20.50 19.47 19.37 19.02 Palmitic 11.16 9.42 9.11 9.90 Oleic 14.40 11.85 11.53 12.64 Linoleic 0.12 0.11 0.10 0.07 Since VCO is mostly made of saturated MCFA, it do not oxidize, stable and can not be harmed by free radicals. VCO which is high in lauric acid (C-12) (typically 46%-49%) and caprylic (C-10) and myristic (C-14) acid. The monoglyceride of lauric acid, which the body makes when lauric acid is ingested, kills cytomegalovirus and pathogenic bacteria. Lauric acid is so disease fighting that it is present in breast milk (Enig, 1993). The body converts lauric acid to a fatty acid derivate (monolaurin), which is the substance that protects infants from viral, bacterial or protozoal infections. It was showed that monolaurin has virucidal effects on RNA and DNA viruses, which are surounded by a lipid membrane. It was reported that MCFA,such as lauric acid have adverse effects on other pathogenic microorganisms, including bacteria, yeast and fungi. These fatty acids and their derivate actually disrupt the lipid membranes of the organisms andthus inactivate them (Issacs and Thomar 1991 ; Issacs et al. 1992 ; Hierholzer and Kabara, 1982). As shown on Tabel 3, the VCO was produced with isolate of Latobacillus plantarum showed the highest of laurat acid (48,76%) followed by Candida rugosa(47,86%) and Aspergillus oryzae (41.56%). Table 4 showed analysis of triglyceride content in VCO were produced by the method was mentioned above. We expected to find the product with a low content of triglyceride as everybody hopes in order to get healthier condition after they consuming VCO. The result showed that the VCO produced by using the stater of K3A exhibited the lowest triglyceride content, followed by VCO by using K2A starter and VCO by using K1A starter. The VCO were produced through enzymatic fermentation contained low free fatty acid and moisture content (0.02-0.03%). It has visual appearance of water clear, good smell and long lifetime. It was assumed to be have more than 1 year storaging. We have found that our tested VCO product contained moisture of approximately 0,06-0,08%, while the highest showed by the control indeed that was 0,12%. This was due to the extracting system which was depended on filtering process using a filter paper only via gravitation. To have more decreased of moisture content, it is recommended to use evaporation process by using such as a water bath system. Conclusions The VCO that was produced by enzymatic fermentation process using starter of Lactobacillus plantarum. contained high lauric acid and low triglyceride content. Present of lauric acid in diet is very important for health since it was reported that has proven to be preventative substance for heart disease, cancer, degenative condition or infectious disease. Tabel 4 Analysis of triglyceride and moisture content of VCO using solid starter of isolates K1A, K2A, and K3A. No Product of VCO Triglyceride (IU/ml) Unit Methods Moisture content (%) 1 K1A solid starter 0.62 IU/ml Specrofotometri 0.06 2 K2A solid starter 0.52 IU/ml Spectrofotometri 0.06 3 K3A solid starter 0.47 IU/ml Spectrofotometri 0.08 7 Control 0.58 IU/ml Spectrofotometri 0.12 651

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