Scholarly article on topic 'Production of Human Milk Fat Replacement Rich of 1,3-dioleoyl-2-palmitoilglycerol From Enzymatic Interesterification Tripalmitin, Ethyl Oleate And Mixture of VCO, Soybean Oil And Fish Oil'

Production of Human Milk Fat Replacement Rich of 1,3-dioleoyl-2-palmitoilglycerol From Enzymatic Interesterification Tripalmitin, Ethyl Oleate And Mixture of VCO, Soybean Oil And Fish Oil Academic research paper on "Agriculture, forestry, and fisheries"

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Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — Sadiah Djajasoepena, O. Suprijana, R. Saadah Diana, Uji Pratomo, Sisca Puspitasari

Abstract Human Milk is naturally the only source of food for infant in their early life. It contains 2-6% lipid which provides about 50% of the total energy needed by the infant. Human milk fat (HMF) mainly as TAG with the specific fatty acid composition, palmitic acid (C16:0) (20-25%), which is primary located at sn-2 of glycerol bonds (70%) and oleic acid (C18:1), located at sn-1,3 (35%). HMF also provide fatty acids such as linoleic acid, linolenic acid, EPA, DHA and lauric acid that are very important for infant. The purposes of this research are to synthesize of 1,3-dioleoyl-2-palmitoilglycerol (OPO) and to determine the best composition of OPO, VCO, soybean oil and fish oil for HMFS production for infant formula. Interesterification of tripalmitin and ethyl oleate using immobilized lipase from Rhizomucor miehei (Lipozym RM IM) were used to synthesize of OPO. Interesterification product of mixed VCO, soybean oil and fish oil that are source of lauric acid, linoleic acid, α-linolenic, EPA and DHA, were formulated in mass ratio (58:20:20:2) and (70:18:10:2) for obtaining HMFS which have fatty acids composition similar or close to HMF. Composition of fatty acids from product were analyzed by GCMS. From this research, were obtained HMFS containing palmitic acid as much as 28.89% where 84.49% of that are located at sn-2 while sn-1,3 position are dominated by oleic acid as much as 55.11% from the total 38.7% and 70:18:10:2 w/w is the best composition of interesterification product, VCO, soybean oil and fish oil to obtain HMFS similar to HMF.

Academic research paper on topic "Production of Human Milk Fat Replacement Rich of 1,3-dioleoyl-2-palmitoilglycerol From Enzymatic Interesterification Tripalmitin, Ethyl Oleate And Mixture of VCO, Soybean Oil And Fish Oil"

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Procedía Chemistry 16 (2015) 384 - 391

International Symposium on Applied Chemistry 2015 (ISAC 2015)

Production ofHuman Milk Fat Replacement Rich of l,3-dioleoyl-2-palmitoilglycerol From Enzymatic Interesterification Tripalmitin, Ethyl Oleate And Mixture of VCO, Soybean Oil And Fish Oil

Sadiah Djajasoepena*, O. Suprijana, Saadah Diana R., Uji Pratomo, and Sisca Puspitasari

Department of Chemistry, Padjadjaran University, Jl. Raya Jatinangor, Km. 21, Sumedang, Indonesia.

Abstract

Human Milk is naturally the only source of food for infant in their early life. It contains 2-6 % lipid which provides about 50% of the total energy needed by the infant. Human milk fat (HMF) mainly as TAG with the specific fatty acid composition, palmitic acid (C16:0) (20-25%), which is primary located at sn-2 of glycerol bonds (70%) and oleic acid (C18:l), located at sn-1,3 (35 %). HMF also provide fatty acids such as linoleic acid, linolenic acid, EPA, DHA and lauric acid that are very important for infant. The purposes of this research are to synthesize of l,3-dioleoyl-2-palmitoilglycerol (OPO) and to determine the best composition of OPO, VCO, soybean oil and fish oil for HMFS production for infant formula. Interesterification of tripalmitin and ethyl oleate using immobilized lipase from Rhizomucor miehei (Lipozym RM IM) were used to synthesize of OPO. Interesterification product of mixed VCO, soybean oil and fish oil that are source of lauric acid, linoleic acid, a-linolenic, EPA and DHA, were formulated in mass ratio (58:20:20:2) and (70:18:10:2) for obtaining HMFS which have fatty acids composition similar or close to HMF. Composition of fatty acids from product were analyzed by GCMS. From this research, were obtained HMFS containing palmitic acid as much as 28.89% where 84.49% of that are located at sn-2 while sn-1,3 position are dominated by oleic acid as much as 55.11% from the total 38.7% and 70:18:10:2 w/w is the best composition of interesterification product, VCO, soybean oil and fish oil to obtain HMFS similar to HMF.

©2015Published byElsevier B.V. Thisisanopenaccess article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of Research Center for Chemistry, Indonesian Institute of Sciences Keywords: Human milk fat replacement; OPO; Lipozym RM IM

Nomenclature

g = gram

* Corresponding author. Tel.: +62818624735 E-mail address: sadiah@unpad.ac.id

1876-6196 © 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of Research Center for Chemistry, Indonesian Institute of Sciences doi: 10.1016/j.proche.2015.12.068

mL = milliLiter °C = degree Celsius

1. Introduction

Milk (ASI) is the food at the same time a very important beverage for infants from birth until the beginning of the age of six months. ASI provides a lot of content that is essential for the growth and development of the baby, both for his endurance and for the development of the brain, such as protein, fat, carbohydrates, vitamins and hormones. Therefore, the baby is in dire need of breast milk as the primary nutrients during the early birth.

At the present time many babies who are not able to enjoy the milk from its mother due to several things, for example mothers can not breastfeed because of work, suffering from a disease that causes the mothers are not allowed to breastfeed their babies, some are due to the mother died, and the reason - the reason Other causes of the baby can not get milk from their mothers. This causes the infants consuming formula kandunganya not as perfect as breast milk, so that the necessary formula implies that at least similar to human milk.

One of the most important ingredient in breast milk is fat. The fat contained in milk, as much as 2-6%, which provides 50% of the total energy needed by infants, therefore breast milk is the main energy source for the baby. As many as 98% of the milk fat is in the form of triacylglycerols (TAG) with specific fatty acid composition. Fatty acids contained in breast milk which are palmitic acid (16: 0) and oleic acid (18: 1). Palmitic acid contained in the milk fat as much as 20-25% with 64% of the total are in the sn-2 position in order to TAG \

Most infants who are not breast fed from his mother, consuming milk with a fat content of certain formula (Human Milk Fat Substitute) derived from vegetable oils or dairy. Many HMFS containing fatty acid composition similar to fatty acids in breast milk, but differ in structure intramolekularnya 2. TAG from the vegetable oil contains palmitic acid which plays on sn-1.3 in order gliserolnya.

Palmitic acid which is positioned at the sn-glycerol 1.3 in order to be hydrolyzed by the enzyme lipase in the intestine into free fatty acids (Free Fatty Acid). The free fatty acids would bind calcium in the gut and changed to 'calcium soaps "that are insoluble. Formation of "calcium soaps" insoluble this would cause a loss of energy in the stool and just as with the loss of calcium that can affect bone mineralization. In addition to the stretcher, the high formation of "calcium soaps" insoluble can cause constipation, a condition that is not good for the baby 2.

A study has observed that the palmitic acid sn-2 position in the TAG can reduce the formation of "calcium soaps" insoluble. Therefore, the position of palmitic acid in TAG HMFS very important for the process of absorption of fat and calcium 3.

l,3-Dioleoil-2-Palmitoilgliserol (OPO) TAG is a structure that is important in toddler nutrition. Breast milk contains palmitic fatty acids which is dominant in the sn-2 position of the TAG different from HMFS from vegetable oils or dairy cows are at the sn-1,3. OPO can be produced by interesterification reaction with the help of specific lipase at the sn-1,3 positions to release palmitic acid from tripalmitin which is its substrate in the sn-1 and sn-3 and later replaced by oleic acid.

Breast milk fats also contain essential fatty acids obtained from the nutritional intake of the mother, the fatty acids including linoleic acid (18: 2 ®-6) and linolenic fatty acid (18: 3 ®-3). Breast milk fat also mengadung AA and DHA from 0.5 to 2.0%, its level depends on the eating habits of the mother. AA and DHA are essential fatty acids in membranes also for bone mass and bone mineral caused by the influence of fatty acid biosynthesis of prostaglandins. DHA is connected to the development of brain and visual system. Thus, infant formulas must contain LCPUFA (long-chain polyunsaturated fatty acids) in an amount similar to breast milk 4.

On the basis of these explanations is needed formulations fat content is OPO, linolenic acid, linoleic acid, EPA (eikosapentanoat acid) and DHA (acid dokosaheksanoat) and lauric acid in infant formula is similar to breast milk, so the content of the formula is similar to breast milk. The contents can be obtained from fish oil (EPA and DHA), Virgin Coconut Oil (lauric acid) and soybean oil (linoleic acid and a-linolenic acid).

The purpose of this research are to producing OPO of tripalmitin and ethyl oleic through enzymatic interesterification reaction using the enzyme lipase from Rhizomucor miehei (Lipozyme RM IM) and determine the best composition of the OPO, VCO, soybean oil and fish oil for the production HMFS which implies similar to breast milk fat.

2. Materials and Equipment

2.1. Material

Materials used in this study is Virgin Coconut Oil (VCO), soybean oil and salmon oil is also used Lipozyme RM IM which is the enzyme lipase from Rhizomucor miehei which diamobilkan in acrylic resin and porcine pancreatic lipase type of Vis.

2.2. Chemicals

Chemicals used include ethyl oleate, tripalmitin, 2.7-diklorofluorosens, silica gel G 60 F254, bile salts, tris HC1 buffer (pH 8), sodium chloride, sodium sulfate, toluene, methanol and sulfuric acid. The solvent used is n-hexane, diethyl ether, formic acid, acetic acid and ethanol.

2.3. Equipment

The equipment used in this study is a rotary evaporator and water bath shaker. Tools for product analysis that Gas Chromatography Mass Spectroscopy (GCMS) and glassware commonly used in biochemical laboratories.

3. Research Methods

3.1. Production OPO through enzymatic interesterification reaction

Tripalmitin (PPP) as much as 4 grams of ethyl oleate is mixed with the 250 mL Erlenmeyer flask at a mole ratio of 1: 6. Then added the enzyme lipase (Lipozyme RM IM) as much as 10% of the total number of mole ratio of tripalmitin (PPP) and ethyl oleate. The mixture was reacted in a shaker water bath at 200 rpm at a temperature of 500C for 2 hours. Furthermore, the enzyme lipase is separated from the mixture by filtration using filter paper.

3.2. Production of Human Milk Fat Subtitutes (HMFS)

Interesterification products mixed with coconut oil, soybean oil and fish oil in the Erlenmeyer flask on a total mixture of 4 grams in each weight ratio (58: 20: 20: 2 w/w) and (70: 18: 10: 2 w/w). Each mixture is then stirred using a magnetic stirrer.

3.3. Analysis of fatty acid content in the HMFS product

Each of these mixtures (HMFS) weighed as much as 20 mg, then put into a test tube closed. Then added 1 mL of toluene and 2 ml of 5% sulfuric acid solution in methanol. The mixture was incubated at a temperature of 500C for 24 hours. The mixture was then added a solution of 5% sodium chloride as much as 5 mL and then extracted by n-hexane 5 mL 2 times. Non-polar layer (top) was added sodium sulfate and then decanted and then separated from the solvent using a rotary evaporator until the volume it occupies ± 1 mL and analyzed using GC-MS.

3.4. Analysis of HMFS regiospecific

3.4.1. Hydrolysis TAG in HMFS

HMFS mix that has the best composition weighed as much as 0.1 grams and then put into a test tube and add tris HC1 buffer pH 8 in 1 mL, 0.05% bile salts in distilled water (w / v) of 0.25 mL, 2.2% calcium chloride in distilled water (w / v) of 0.1 mL and 0.25 mg of pancreatic lipase, test tube containing the mixture is then incubated in a water bath for 3 minutes at a temperature of 400C and then added 1 mL of 6 M HC1 and 1 mL of diethyl ether and incubated for 15 minutes at a speed of 40 rpm. Then centrifugation supernatant results in evaporation.

3.4.2. Separation hydrolysis using TLC

Products of hydrolysis spotted on a plate measuring 20 cm x 20 cm that had been coated with silica gel G 60 F254, the developer solvent hexane: diethyl ether: acetic acid (50: 50: 1 v / v). TLC plate eluted in a container that has been saturated with solvent developers to reach the limit on the TLC plate. Noda formed then viewed under UV light at X = 254 nm. MAG stain scraped and dissolved by n-hexane and then centrifuged. The results of centrifugation supernatant is evaporated using a rotary evaporator, to obtain solvent-free samples.

3.4.3. Analysis of hydrolysis products using GCMS

TLC yield components weighed 20 mg, put in a test tube with a lid, dissolved in 1 mL of toluene and 2 ml of 5% sulfuric acid in methanol. The mixture was incubated at a temperature of 500C for 24 hours. The mixture was added 5 mL of 5% sodium chloride solution and extracted with 5 mL of n-hexane twice, nonpolar layer was added sodium sulfate. Nonpolar layer (fatty acid methyl ester) pipette and evaporated the solvent through a rotary evaporator to a volume of about kiral mL and analyzed through GCMS

3.5. Calculation of fatty acids at position sn-1.3

Fatty acids in the sn-1,3 calculated by: sn-1,3 (%) = (3T - sn-2) / 2. Where T is the total content of fatty acids present in the sample.

4. Results And Discussion

4.1. Production OPO Through Enzymatic interesterification reaction

OPO production is done by enzymatic interesterification reaction between tripalmitin and ethyl oleate using the enzyme lipase from Rhizomucor miehei (Lipozyme RM IM) to the reaction conditions based on the results of research5.

Tripalmitin and ethyl oleate in a mole ratio (1: 6) were mixed in Erlenmeyer flask is then reacted in a water bath shaker for 2 hours at a temperature of 500°C at a speed of 200 rpm. Interesterification reaction between tripalmitin and ethyl oleic happen with the help of the enzyme lipase from Rhizomucor miehei (Lipozyme RM IM) were added to the flask as much as 10% of the total weight of the mixture of substrates. The temperature used in the reaction is the optimum temperature of the enzyme lipase which is 500C.

Tripalmitin and ethyl oleate which is a substrate, enzymatic interesterification reactions. According to the research 6, the exchange interesterification reaction is a reaction of two ester groups of different esters. Excess enzymatic interesterification is milder processing conditions, damage to unsaturated fatty acids can be suppressed, and the exchange of acyl group can be controlled7.

Tripalmitin which is a TAG is also an ester will exchange groups in the form of fatty acid ester is palmitic acid is located at position sn-1 and sn-3 with oleic acid ethyl ester in the form of oleic to produce l,3-dioleoil-2 -palmitoilgliserol. Interesterification reaction is an enzymatic reaction that occurs with the aid of a lipase to produce products with a distribution of molecular species that are more selective. Lipase from Rhizomucor miehei (Lipozyme RM IM) is an enzyme that is specific to the position sn-1 and sn-3 that can hydrolyze tripalmitin at these positions so that oleic acid can replace the position of palmitic acid in position sn-1 and sn-3 to produce OPO ,

Enzymatic interesterification reaction in this study conducted without a solvent (solvent free system) to restrict the mass transfer. In addition to the condition without solvent was also done because the substrate is in liquid form (oil) with a sufficiently high temperature is 500°C, which also carry out the reaction HMFS interesterfikasi for the production of vegetable oil mixture 8.

4.2. Production HMFS

HMFS (Human Milk Fat Substitute) are fats that are used as a fat substitute that is inserted into the breast milk formula. OPO is TAG is predominantly found in breast milk fat, therefore in this study, OPO is produced as one of the constituent HMFS. There are many other fatty acids have a very important role even though the amount is not so dominant in the milk fat. The fatty acids include linoleic acid (CI 8: 2 ®-6), a- linolenic acid (C18: 3 ®-3), EPA (C20: 5 ©-3), DHA (C22: 6 ®-3 ) and lauric acid (C12: 0).

EPA and DHA are long-chain fatty acids with a double bond compound or so-called LCPUFA (Long Chain Poly Unsaturated Fatty Acid) and omega 3. Epidemiological studies have shown that EPA and DHA have several health benefits associated with heart disease, kidney disorders, immunity, inflammation, diabetes and allergies. DHA is a fatty acid as the dominant constituent of the brain and retinal tissue membranes and plays a complex role in the development of nerve cell function (brain), photoresepsi (light reception) in infants, especially premature infants and inflammatory diseases 9. Laurie acid contained in the milk fat as much as 9.4%. Laurie acid can increase metabolism, kill viruses, bacteria and protozoa.

Production HMFS containing OPO and other essential fatty acid linoleic acid, a-linolenic acid, EPA, DHA and lauric acid made by mixing interesterification products containing OPO, virgin coconut oil (VCO), soybean oil and salmon oil on variations weight ratio 58: 20: 20: 2 and 70: 18: 10: 2 in the bottle vial with 4 grams total mixture.

VCO is used as a source of lauric acid, because VCO contains a lot of lauric acid which is about 45%. VCO contains lauric acid which is used as much as 51.23%.

Soybean oil is used as a source of linoleic acid and a-linolenic acid. Soybean oil is the right material to be used as a source of linoleic acid and a-linolenic acid, because it contains high linoleic acid is as much as 50% while the a-linolenic acid contained as much as 5.7%.

Fish oil is a source of ®-3 fatty acids (EPA and DHA). Fish oil that is used in this study is from salmon fish oils containing 12.31% DHA and 9.23% EPA.

4.3. Analysis of Total Fatty Acid Content in Products HMFS Using GCMS

Analysis of fatty acid content in the product HMFS performed using methods GCMS. This techniques can be used to determine fatty acid composition of lipid in full in a very fast time. GCMS is a method that combines gasliquid chromatography and mass spectroscopy to identify different substances in a test sample. Because of this, the fatty acid components of lipids to be converted into a simple derivatives are more volatile, normally is converted into methyl ester 10.

Derivatization process is done through mixing products HMFS with toluene and 5% sulfuric acid in methanol and then incubated for 24 hours at a temperature of 500°C. Sulfuric acid serves as a catalyst in the reaction. After incubation process is completed to the mixture was added sodium chloride and extracted by n-hexane. This extraction serves to pull the product fatty acid methyl esters in non-polar by n-hexane non-polar as well. Fatty acid methyl esters are separated from the solvent n-hexane using a rotary evaporator until approximately 1 mL.

From the analysis using GCMS, chromatograms obtained for a mixture of I and II. From the chromatogram obtained composition of fatty acids in the mixture are shown in Table 1 below.

Table 1 Composition of fatty acids in the product mix HMFS from OPO, VCO, soybean oil and fish: I mixture (58: 20: 20: 2)

and II (70: 18:10: 2) w/w were analyzed using GCMS.

Fatty Acid OPO The content of HMFS /% y^Q Soybean Salmon oil oil Mixture I (%) Mixture II (%) HMFa (%)

Caprylic Acid (C8: 0) 5,99 0,49 0,96

Capric Acid (C10: 0) 7,49 0,88 0,82

Laurie Acid (C12: 0) 45,83 4,29 6,77 8,86 ±3,72

Myristic Acid (C14:0) 18,89 0,1 3,79 3,40 10,05 ± 3,02

Palmitate Acid (C16:0) 19,87 11,94 29,17 23,73 28,89 26,86 ± 2,71

Stearic Acid (C18:0) 2,77 15,9 3,18 2,96 4,09 ± 0,67

Oleic Acid (CI8: 0) 70,82 7,02 43,60 38,72 30,82 ± 4,07

Linoleic Acid(C18: 2) 50,53 0,85 11,42 8,84 ± 1,24

Linolenic Acid (C18: 3) 3,87 0,15 0,13 0,3 ±0,14

EPA (C20: 5) 12,31 0,27 0,27 0,21 ±0,11

DHA (C22: 6) 9,23 0,15 0,18 0,9 ± 0,29

This table show that both products HMFS resulting mixture has a composition of fatty acids approaching the composition of fatty acids in HMF. The fatty acids include lauric acid, palmitic acid, oleic acid, linoleic acid and linolenic acid. Laurie acid is contained in the mixture I as much as 4.29%, while in the mixture II contained more as many as 6.77%. The second number is still smaller than the amount of lauric acid contained in HMF is equal to 8.86 ± 3.72%, but the content of lauric acid in the mixture II is still in the range of lauric acid content in the HMF, the results of the analysis II we concluded that the mixture is a mixture that contains lauric acid most closely with content in HMF.

Other fatty acids are palmitic acid contained as much as 23.73% in the mixture I, whereas in the mixture II contained 28.89%. In HMF, palmitic acid contained as much as 26.86 ± 2.71% so that the amount of palmitic acid in the mixture II is still in the range of the amount of palmitic acid in the HMF, while the numbers in the mix I was still

lower than the amount in the HMF. From this analysis we concluded that the mixture II is a mixture that contains palmitic acid comes closest to the content in HMF.

Oleic acid is contained in the mixture I as much as 43.60%, while in the mixture II smaller in number as many as 38.72%. There is a discrepancy in the value of both, which should have been the amount of oleic acid in the mixture II should be greater than the amount in the mixture first because the number of OPO is added to the mixture II more than in the first mixture, ie the mixture II OPO added by 70% while in I mix only by 58%. This can occur when the process is possible because the methylation derivatization or mixture II, not all of which are oleic acid in the mixture is esterified to methyl esters of fatty acids so that when the analysis is done using GCMS fatty acid was not detected because they have a high boiling point, whereas one of the main requirements of a compound to be analyzed through GCMS are to be volatile. However, both the amount of oleic acid content in the mixture, the content of oleic acid in the mixture II is the number closest to the number in the HMF compared with a mixture I. In mixture II contained of oleic acid as much as 38.72%, which a maximum amount of oleic acid in the HMF is equal to 34.89%, while the amount of oleic acid in the mixture I is 43,60%.

Other essential fatty acids are linoleic acid contained as much as 0.85% in the mixture I and as much as 11.42% in the mixture II. Even this analysis results similar to those seen in oleic acid, which should mix I contains linoleic acid is higher than the mixture II due to the mix I, soybean oil which is a source of linoleic acid added as much as 20%, while the second is added to the mix as much as 18% , This can happen as well as that occurred in oleic acid, linoleic acid that is not all that is contained in the first mixture is converted into methyl esters are more volatile than in the form of fatty acids. From the results of this analysis, it is concluded that the mixture II contains linoleic acid that comes closest to HMF. Value 11.42% had a difference of 1.34% with a maximum amount of linoleic acid in the HMF is equal to 10.08% as linoleic acid content in the HMF is equal to 8.84 ± 1.24%.

Other fatty acids derived from soybean oil are linolenic acid. The fatty acids contained as much as 0.15% in the first mixture, and as much as 0.13% in the mixture II. Both values are still lower compared to the amount of linolenic acid in the HMF as many as 0.3± 0.14%.

Omega-3 fatty acids are EPA and DHA derived from fish oil, which contains quite similar to HMF. In a mixture of I and II contained as much as 0.27% EPA. This amount is in the range of EPA content in HMF is equal to 0.21 ± 0.11%. The amount of EPA in the mix I and II arejust as much, because fish oil is added to the mix I and II as much. From these results concluded that neither I nor II mixture, the mixture is approaching HMF in terms of the content of EPA.

DHA is contained in the mixture I as much as 0.15%, while in the second mixture contained as much as 0.18%. The second number is still lower than the amount in the HMF is equal to 0.9 ± 0.29%. The difference between the content of both products produced with HMF is large enough, both of which do not fit into the range of the content of DHA in HMF, whereas DHA is an ®-3 fatty acid that is essential for brain development of the baby.

From the foregoing it is concluded that the mixture II is HMFS products that contain fatty acids that are more similar to HMF as compared to the first mixture, because the fatty acids contained in the mixture II has a number that is more similar to HMF is lauric acid, palmitic acid, acid oleic, linoleic acids, EPA and DHA. While the mixture of fatty acids that Ijust linolenic acid and EPA are closer to HMF fatty acids in comparison to the mixture II.

Mixture II and then analyzed further that regiospesifik analysis to determine the type and amount of fatty acid sn-2 position on the framework TAG.

4.4 Analysis of Regiospecific HMFS Product 4.4.1 Pancreatic Lipase Enzyme Hydrolysis TAG

Analysis of fatty acid distribution in TAG conducted through analysis by means of hydrolyzing TAG regiospesifik into 2-monoacylglycerol (2-MAG) and two free fatty acids using pancreatic lipase.

Regiospesifik analysis was conducted to determine the type and amount of fatty acids that occupy the sn-2 position in glycerol skeleton constituent TAG. From this analysis are expected to palmitic acid is the predominant fatty acid in that position is about ~ 70% as well as on fat milk contains as much as 20-25% palmitic acid and 70% -it is at the sn-2 position of the TAG frame \

HMFS added products of porcine pancreatic lipase, tris-HCl buffer (pH 8), bile salts and calcium chloride. Pancreatic lipase function as biocatalysts. The use of pancreatic lipase regiospesifik analysis is to hydrolyze TAG into 2-MAG and free fatty acids. In accordance with the fats contained in milk, fat will be absorbed by the colon is

in the form of MAG and free fatty acids instead of TAG. Pancreatic lipase along with bile salts hydrolyzing TAG. Pancreatic lipase is an enzyme that has an optimum pH from 7.5 to 8, so that added tris-HCl buffer (pH 8) to keep the pH in pancreatic lipase. The mixture was incubated for 3 minutes at a temperature of 400°C. Heating function is to activate pancreatic lipase enzymes for optimum working temperature of 400°C.

The reaction mixture was extracted by diethyl ether, to form two phases. Nonpolar phase which is located at the top of the mixture is separated. Diethyl ether is a nonpolar compound that will bind the lipid mixture which is also a non-polar compounds, according to pinsip like dissolved like, a compound will dissolve in the solvent that has a polarity that is relatively the same level. The reaction results in the form of a lipid mixture is separated from the solvent using a rotary evaporator.

4.4.2 Separation of 2-MAG through Preparatif Thin Layer Chromatography (TLC)

TLC a chromatographic method that is used to separate mixtures of compounds from a small scale. At TLC analysis used stationary phase is silica gel G 60 F254 diluted by distilled water with a ratio of silica gel: distilled water is 1: 2 (g / mL). Silica gel which has a homogeneous then superimposed above 1 mm thick glass plate, then dried in the open air. Silica gel is then activated in an oven at a temperature of 100°C for 1 hour to remove moisture remaining in the silica, so that more optimal separation of the compounds. The presence of water in silica may inhibit the rate of elution, because the samples were separated in the form of fatty acid nonpolar while water is polar.

Products of hydrolysis spotted over a silica plate then eluted in a chamber that has been saturated by the developer solvent that is a mixture of n-hexane, diethylether and acetic acid (50:50:1 v/v/v).

Results elution then viewed under UV light at X = 254 nm. In the four stains visible license plate. Stains were eluted farthest row is ethyl esters, TAG, fatty acids and 2-MAG bebeas. 2-MAG elute closest comparison to free fatty acids and TAG 10. This is because, MAG has the most polar properties between free fatty acids and the TAG so elute closest to the polar plate (Silica gel).

Noda in the form of 2-MAG immediately scraped to prevent terdekomposisinya 2-MAG for too long in contact with the adsorbent. Silica which has been scraped off and then extracted by n-hexane to attract MAG are still bound to the silica. N-hexane nonpolar be binding MAG non-polar accordance with principles like dissolve like. Centrifugation performed to separate n-hexane containing 2-MAG of silica gel.

Before it is injected into the tool GCMS, 2-MAG first converted into fatty acid methyl ester to be more volatile in the same way the previous HMFS derivatization products.

4.4.3 Analysis of 2-MAG through GCMS

Constituent fatty acid 2-MAG result of hydrolysis were analyzed womb through GCMS. GCMS chromatogram analysis results are shown in Appendix 4.2 that shows peaks representing each of the fatty acids are at the sn-2 position of the TAG framework.

Table 2 shows the type and amount of fatty acids in the position sn-2 and sn-1.3 were analyzed using GCMS. The amount of fatty acid in the sn-1,3 positions can be calculated by the formula:

sn-1,3 (%) = (3T - sn-2) / 2 where T is the total amount of fatty acids in HMFS.

In Table 2 shows that the types of fatty acids that are most dominant in the sn-2 position of the TAG framework in HMFS is palmitic acid product that is as much as 73.24%. Other fatty acids are in a very small amount is lauric acid (1.37%), myristic acid (0.76%), stearic acid (0.74%), oleic acid (6.91%) and linoleic acid (0,32 %). Of the total amount of palmitic acid in the product that is equal to 28.89%, 84.49% of which position sn-2 in the framework of TAG. This indicates that the content of this HMFS approaching HMF content, which according to research Jimenez et al. (2009) contained palmitic acid in the milk fat is equal to 20-25%, 70% of them are in the sn-2 position in the framework of its TAG. The more abundant palmitic acid which is distributed at the sn-2 position of the formation of calcium soap will also decrease. Calcium soap is a substance that is not good for babies because it can interfere with bone mineralization process and cause constipation in infants. Calcium soap formed from calcium binding reaction inside the body of the baby by palmitic acid free. Free palmitic acid is derived from the hydrolysis reaction of one molecule of TAG by pancreatic enzymes in lipid metabolism process that produces one molecule of 2-MAG and two molecules of free fatty acids. If the distributed palmitic acid at the position sn-1,3 then hydrolysis TAG will produce two molecules of palmitic acid free will bind calcium and form calcium soap, otherwise if palmitic acid is distributed

at the sn-2 position of TAG hydrolysis eating will produce the 2-palmitoilgliserol more easily absorbed by the intestine compared to palmitic acid in free form.

Table 4.2 Type and Number of Fatty Acids In sn-2 position and sn-1.3 were analyzed through GCMS.

Asam Lemak Contain / %

Total sn-2 sn-2a sn-1,3

Laurie acid (C12: 0) 6,77 1,37 6,74 6,78

Myristic acid (C14: 0) 3,40 0,76 7,40 1,40

Palmitateacid(C16: 0) 28,89 73,24 84,49 1,1

Stearic acid (C18: 0) 2,94 0,74 8,37 0,23

Oleic acid(C18: 1) 38,72 6,91 5,94 55,12

Linoleic acid (C18: 2) 11,42 0,32 0,93 16,67

' = amount of fatty acid in the sn-2 position of the total amount of fatty acids in the HMFS is calculated by an equation [sn-2 Fas x 100 / (3 x total FAS).

In Table 2 also shows that the most predominant fatty acid in sn-1,3 position is oleic acid which is as much as 55.11%, this number is higher than in the HMF content is 30.5%. Regiospecific From the analysis it can be concluded that the distribution of fatty acids in the product position HMFS approaching HMF, where 84.49% of the total palmitic acid as much as 28.89% in products that are in the sn-2 position, while the position sn-1.3 dominated by oleic acid is as much as 55.11%. This can reduce the formation of calcium soap which can interfere with the process of bone mineralization and absorption of fat in infants.

5. Conclusion

From this study we concluded:

1. OPO can be produced through enzymatic interesterification reaction of tripalmitin and ethyl oleate using lipase from Rhizomucor miehei (Lipozyme RMIM) with the acquisition, in the framework of TAG, as much as 84.49% palmitic acid sn-2 position, while at the sn-1 position, 3 occupied oleic acid as much as 55.11% of the total.

2. Best Composition interesterification proceeds, VCO, soybean oil and fish oil to produce HMFS who have fatty acid composition similar to HMF in this study was 70: 18: 10: 2 w / w.

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