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nzymatic glycerolysis for conversion of sunflower oil toood based emulsifiers
alaya K. Naika, S.N. Naika,∗, Sanat Mohantyb
Center for Rural Development & Technology, Indian Institute of Technology, Hauz Khas, Delhi, IndiaDepartment of Chemical Engineering, Indian Institute of Technology, Hauz Khas, Delhi, India
r t i c l e i n f o
rticle history:eceived 8 August 2013eceived in revised form1 November 2013ccepted 14 November 2013vailable online xxx
eywords:
a b s t r a c t
High temperature glycerolysis widely used in industrial scale for production of food-based emulsifiershas many disadvantages. On the other hand, high cost, unavailability, low activity and high reactiontime of enzymes limit the use in industrial scale glycerolysis reactions for the production of food-basedemulsifiers (i.e. monoglycerides). The goal of this project is to develop an efficient and optimal glycerolysisprocess for production of monoglycerides. This study found that optimal production through enzymaticglycerolysis was achieved with 15 wt% loading of Fermase CALB 10000 and oil to glycerol molar ratio wasoptimized at 1:5. A tert-butyl alcohol system was developed carefully with evaluation of more than 10
mulsifiersonoglycerides
ermase CALB 10000
organic solvents. The suitable co-solvent addition was 1:50 molar ratio with respect to oil. In the above-optimized condition, the conversion of monoglyceride was 50–60% at 2–3 h and 70–80% at 5 to 6 h ofreaction time. The reaction time was optimized at 5 h. Subsequent to the following reaction was foundthat, there is no drastic reduction of enzyme activity and could be reused for next reaction. Due to thisbehaviour, we recommend Fermase 10000 as an effective and low cost enzyme for future glycerolysisreaction in industrial scale.
Global industry analysts have released a comprehensive reportn the emulsifiers market [1]. Growing use of emulsifiers in foodnd beverage and personal care products, rising popularity of nat-ral emulsifiers, the growing trend of fat replacement in foodroducts and continuous product innovations are currently driv-
ng the global market for emulsifiers. Driven by these robust growthactors, the market is primed to reach volume sales of 2.6 million
etric tons by the year 2017 [1].The requirement of emulsifier increases as the monoglycerides
MG) mixtures with diglycerides (DG) account for approximately5% of the emulsifier production and have various applications inifferent fields. In the food industry, MG is widely used in bakeryroducts, margarines, dairy products, and confectionary because ofheir emulsifying, stabilizing, and conditioning properties. They arelso important in cosmetic and pharmaceutical industries as drugarriers and for consistency improvements in creams and lotions.
Please cite this article in press as: M.K. Naik, et al., Enzymatic glycerolysis f(2013), http://dx.doi.org/10.1016/j.cattod.2013.11.005
wing to their lubricating and plasticizing properties, MG is alsosed in textile, fiber processing as well as in the production oflastics [2].
As per “World Health Organization” instruction emulsifiers(E.E.C. code: E471) mention that: (a) the content of monoglyceridesand diglycerides must be at least 70% (w/w), (b) monoglyceridescontent must not be <30% (w/w); (c) no more than 3, 7, and 10%of fatty acid, free glycerol, and triglyceride respectively in productssuch as bread, ice cream, and margarine [3].
Emulsifiers can be manufactured by various routes; i.e. (a) glyc-erolysis of triglycerides, (b) hydrolysis of triglycerides, (c) directesterification of glycerol with fatty acids and (d) transesterifica-tion of glycerol with fatty acid methyl esters. In this synthesis, ourfocus is mainly on production of monoglycerides by glycerolysisof triglycerides. The transesterification of fatty acid methyl ester(FAME) with glycerol to produce monoglycerides is an easier route;however, the priority of FAME in biodiesel sector is more importantthan emulsifier production. Hence used for transportation sector.
Commercially, MG is widely manufactured by the glycerolysisof fats or oils. The glycerolysis reaction is accelerated by the use ofinorganic alkaline catalysts, such as NaOH, KOH or Ca(OH)2, at hightemperatures (220–260 ◦C). The MG content in the product variesfrom 10 to 60%, depending upon the reaction condition. ProducedMG is subsequently purified by short-path distillation or molecular
or conversion of sunflower oil to food based emulsifiers, Catal. Today
distillation, to achieve at least 90% purified product [4]. The use ofhigh temperature in the above process leads to the development ofimpurities chemicals that cause burned smell and dark color hav-ing lower value to food. In addition, the high-temperature chemical
Table 1Physico-chemical properties of sunflower oil.
Name
Acid value (mg KOH/g) 0.05–0.58Saponification value (%) 179–185Iodine value (mg I2/100 g) 115–135Unsaponifiable matter (%) <3%Viscosity (cSt) (40 ◦C) 2–2.6Palmitic acid (C16:0) % 5–7Stearic (C18:0) % 2–3
pcdtf
poaoticole
Fomeficrasam
2
2
wm14aobtSpmf
2
gs
rials and methods, 5–20% of enzyme loading were used keeping oil
Oleic (C18:1) % 45–48Linoleic (C18:2) % 44–48
rocess is not suitable for the production of heat-sensitive MGontaining richer in polyunsaturated fatty acid (PUFA). Thus, pro-uction of is heat-sensitive MG has a commercial interest owing toheir nutritional value, which could be further applied as functionaloods, pharmaceuticals additives.
Enzyme-catalyzed glycerolysis reactions are believed to be aotential alternative to the chemical process because requirementf lower temperature (40–60 ◦C). The lower processing temper-ture results in high quality of products. Several investigationsf low-temperature lipase-catalyzed glycerolysis have confirmedhe potential of the enzyme-catalyzed processes, even though anndustrial-scale process is still unfeasible owing to the low effi-iency conversion [5]. The long reaction times, low conversionf reactants, poor miscibility of the hydrophilic glycerol with theipophilic triglecerides at low temperatures and the high cost ofnzymes make the reaction inefficient in commercial levels.
In the glycerolysis reaction, we have mainly focused on the use ofermase CALB 10000 to convert sunflower oil to richer percentagef monoglycerides. In this project, we have used Fermase 10000,anufactured by Fermenta Biotech, Thane Maharashtra, India. This
nzyme is an indigenous product and thus relatively cheap (almostve times cheaper than Novozyme 435) while activity is almostomparable with Novozyme 435. We have tried to optimize theeaction conditions by varying factors like enzyme loading percent-ge, glycerol concentration, type of solvents and concentration ofolvent and reaction time while keeping constant mixing intensitynd temperature. In addition to the experiment, attempted wereade to recover of enzymes, co-solvents and un-reacted glycerol.
. Materials and methods:
.1. Feedstock and chemicals
Sunflower oil (moisture < 0.5%) purchased from the markethile analytical grade of glycerol purchased from Merck, India. Fer-enta Biotech, Maharastra, India graciously donated Fermase CALB
0000 while Novozyme 435 by Novozyme, Denmark. Novozyme35 and Fermase CALB 10000 are both produced from candidantarctica Lipase and are produced as an immobilized productionn acrylate beads. Highly pure (98–99%) scetone, acetonitrile, t-utanol, chloroform, ethanol, n-haxane, n-haptane, methanol, and-pentanol used in these experiments were purchased from Fishercientific, India. HPLC grades tetrahydrofuran, acetone, water, iso-ropanol were purchased from Merck, India. HPLC standards ofonoglycerides, diglycerides and triglycerides were purchased
rom Nu-check prep (USA) and glycerol from Sigma Aldrich, India.
.2. Physico-chemical properties of sunflower oil and glycerol
Please cite this article in press as: M.K. Naik, et al., Enzymatic glycerolysis f(2013), http://dx.doi.org/10.1016/j.cattod.2013.11.005
The fatty acid compositions of sunflower oil were analyzed byas chromatograph. The fatty acid profile, acid value, iodine value,aponification value and unsaponifible matter of the sunflower oil
PRESSay xxx (2013) xxx– xxx
are presented in Table 1. The total moisture content of the reactantmixture before adding enzyme was found in the range 1–2%.
2.3. Enzymatic glycerolysis reaction
Enzymatic glycerolysis reaction was performed in a shaking bedincubator. A 50 ml tightly capped borosil conical flask was for thereaction. It was filled with varying the amounts of oil, enzyme andglycerol as were kept inside in a shaking bed incubator maintainingtemperature at 50 ◦C and shaking speed at 200 RPM. The reactiontime for the vessel was kept for 8 h. The enzyme (Fermase 10000)concentration was varied from 5 to 20 wt%, glycerol concentrationin molar ratio from 5 to 15 wt% with respect to oil. Different co-solvents were used to solubilize glycerol and oil in the reactionmixture.
To study the kinetics of the reaction over the course of the reac-tion, time small volumes of the reaction mixture were withdrawnand filtered through a syringe filled with a cotton wool to removethe enzymes and any other matter before testing. Subsequently,samples were flushed through vacuum distillation to remove theextra solvents. All samples were stored at −20 ◦C prior to analysis.
After the reactions, the enzymes were recovered by the nylonfilter cloth and was washed with 4–5 times with iso-propanol anddried at normal temperature and stored at −20 ◦C for next run.
2.4. Analysis of the reaction mixture
High performance size exclusion chromatography (HPSEC) asused for analysis of triglycerides, diglycerides, monoglycerides andglycerol. The samples collected from the shaking incubator werediluted with tetrahydrofuran (HPLC grade) and filtered by phenex(Phenomenex, Part No: AF0-2207-12) 15 mm Nylon syringe mem-brane filter having pore size 0.2 �m. 20 �l samples were injectedin the HPLC consisting of a refractive index detector with a temper-ature controller maintained at 40 ◦C. Two phenogel columns wereconnected in series, the first is about 100–3000 (50 A) and the sec-ond one is molecules about 500–6000 (100 A) molecular weight,with dimension of 300 × 7.8 mm and a particle size of 5 �m. Themobile phase was tetrahydrofuran and runs of about 30 min, witha 0.7 ml/min flow rate. The analysis of the peak for the HPLC is givenin Fig. 1.
3. Results and discussion
3.1. Effects of various co-solvents
To select the most suitable co-solvent for the glycerolysis reac-tions were carried out by different organic solvents (i.e. acetone,acetonitrile, chloroform, ethanol, methanol, t-butanol, t-pentanol,n-haxane, n-haptane). Those results are mentioned in Fig. 2, whichis agreed with Pawongrat et al. and Yang et al. [7,8] results. Thereis a comparable difference of 20% MG conversion in 5 h of reactiontime with t-pentanol and t-butanol. However, the same reactionscontinued up to 6 h and then up to 8 h and it was found that, there isno such increase of MG percentage with t-pentanol. Thus, consider-ing the higher percentage yield of monoglycerides, it was decidedto carry out the further reactions with t-butanol.
3.2. Effect of enzyme concentration
The percentage of MG formation is a linear relationship with acertain range of enzyme loading. As mentioned in the above mate-
or conversion of sunflower oil to food based emulsifiers, Catal. Today
to solvent and oil to glycerol molar ratio at 50 and 5 respectively.In continue with 5 h of reaction, the conversion of TG to MG by 5%,10%, 15% and 20% were 30%, 33%, 79% and 77% and the same was
of monoglycerides conversion with high solvent ratio. However,after a certain point, there is no further increase of monoglyceridesconversion percentage with respect to the co-solvent addition. The
50
60
70
80
90
nogl
ycer
ides
5% 10% 15% 20%
Fig. 1. HPLC analy
ncreased to 32%, 35%, 80%, 77% in 5.5 h and 33%, 38%, 80%, 78%n 6 h of reaction period (Fig. 3a). From the results, it was foundhat, the conversion of monoglycerides percentage increase withncrease with enzyme loading up to certain range of time shownn Fig. 3b. Comparing with 15% and 20% of enzymatic reaction, theield of monoglycerides is almost similar during 5 to 8 h of reac-ion time. Thus, 5 h of reaction time was taken as optimal periodnd during this period, MG production by 15% and 20% was 79%nd 77%. Though the MG% is depend upon on catalyst loading butt higher concentration enzyme the product conversion decreasesnd this may be due to the active sites of the enzymes moleculesresent in large volume would not be exposed to the substrates,hich is possibly caused by protein aggregation [6]. Thus, finally
5% enzyme loading was chosen for further reaction.
.3. Effect of co-solvent concentration
Please cite this article in press as: M.K. Naik, et al., Enzymatic glycerolysis f(2013), http://dx.doi.org/10.1016/j.cattod.2013.11.005
Addition of co-solvent plays an important role in the productonversion. It increases the mixture homogeneity and stability,hich further help the mass transfer in lower viscosity. More-
ver, more than necessary amount of co-solvent mixing influences
Fig. 2. Effect of different co-solvents compared with %MG conversion.
reaction mixture.
the reactant concentration with respect to the reaction mix-ture, where co-solvent may decrease the rate of reaction as perMichaelies–Menten equation [8]. Therefore, it is important toestablish the optimal solvent dosage in the reaction mixture.
Fig. 4 shows the conversion of monoglycerides with differentmolar ratio (30, 40, 50 and 60) of t-butanol and shows the increase
or conversion of sunflower oil to food based emulsifiers, Catal. Today
10
20
30
40
100 150 200 250 300 350 400 450 500
% M
o
Time (min )
0
10
20
30
40
50
60
70
80
90
0 5 10 15 20
% M
onog
lyce
ride
s
% Enzy me Loading
300 min
360 min
480 min
Fig. 3. (a) Effect of different catalyst concentration compared with %MG conversion.(b) Effect of enzyme loading compared with %MG conversion at specific time period.
Fig. 4. Effect co-solvent addition compared with %MG conversion.
ncrease of solvent concentration from 40 to 50 molar ratios, theres a difference of 17% MG formation at 5 h and 11% MG at 8 h ofeaction, which means very marginal MG formed after 5 h. How-ver, with increase of solvent molar ratio from 50 to 60, there is noubstantial increase (<1–3%) of monoglycerides conversion. Thus, itas decided the co-solvent addition of 50 molar ratios with respect
o oil is optimized condition.
.4. Effect of glycerol concentration
Since the viscosities of oil and glycerol mixture are relativelyigh, the concentrations of the substrate mixture have limitedccess to the enzyme. It may hamper the mass transfer betweenhe substrata to the enzymes; as a result, it may leads to decreasef product conversion. Thus, it is very important to optimize theil to glycerol molar ratios to get higher conversion. By theoreti-ally, the increase of glycerol with respect to oil leads to increasef reactant equilibrium and will be helpful the product conversions per Le-chatelier’s principle. As the same time, glycerol also actss an effective stabilizer against thermal and solvent deactivation2] as well as influence system polarity and homogeneity [8]. Bothnzyme and glycerol are hydrophilic in nature. Thus, the more thanequired amount of glycerol forms a coating around the enzyme,hich subsequently leads to enzyme clumping and deactivation,
s a result lower in monoglycerides conversion [9].
Please cite this article in press as: M.K. Naik, et al., Enzymatic glycerolysis f(2013), http://dx.doi.org/10.1016/j.cattod.2013.11.005
In Fig. 5, the product conversion with respect to oil to glycerololar ratio (1:1, 1:3, 1:5, 1:10, 1:15) are represented. The increase
f glycerol from 1:1 to 1:3 to 1:5, there is increase of monoglyc-rides conversion from 15% to 40% to 79% at 5 h of reaction. The
0
10
20
30
40
50
60
70
80
90
100
100 150 200 250 300 350 400 450 500
% M
onog
lyce
ride
s
Time (min )
1:1 Molar 1:3 Mo lar 1:5 Mo lar1:10 Molar 1:15 Molar
Fig. 5. Effect of glycerol addition compared with %MG conversion.
PRESSay xxx (2013) xxx– xxx
same condition continuing after 6 h, there is no significant increaseof monoglycerides conversion. As the concentration of glycerolincreased from 1:5 to 1:10 to 1:15 molar ratio, there is a significantdecrease of MG formation in the same interval of time. This maybe due to enzyme clumping and inactivation. This results is similarto Yang et al. [8], who found the same trend of results with sun-flower oil:glycerol in 1:4.5 molar ratio in the presence of t-butanol.Pawongrat et al. [7] show optimum monoglycerides formation intuna oil:glycerol at 1:3 molar ratio in the presence of t-butanol.
3.5. Effect of reaction temperature and time
Reaction temperature affects the equilibrium composition ofthe final reaction product. It is reported that at lower reactiontemperature which help the reaction towards production of highcontent of diglycerides [10]. Zhong et al. [11] shows the diglyc-erides content increases with increase of temperature from 35 to45 ◦C in ultrasonic irradiation system; however, a very little differ-ence was observed when reaction temperature was varied from 45to 55 ◦C. This indicates the reaction reached equilibrium and thecomposition at equilibrium would not change within this range oftemperature. Following the above reaction condition, we have triedto fix the temperature at 50 ◦C and optimized the other reactionparameters.
From Figs. 2–5, it can be observed that the conversion of mono-glycerides percentage is increases up to 5 h of reaction. However,to show the behavior of time with conversion ratio, the reactionwas continued up to 8 h. The monoglycerides production was 79%percentages in 5 h of reaction and after that; there is hardly anyincrease of monoglycerides percentage. The difference of mono-glycerides conversion during 5 to 8 h of reaction time is less than2–3%. Keeping interest on economic point of view, 5 h of reactiontime was decided as optimized reaction time.
3.6. Effect of water content
For most lipases, moisture is essential to maintain the enzymeactivity of and allow the formation of an acyl-enzyme complex [7].However, the enzymes prepared from CALB, they can remain highlyactive in a dry state without any water addition [12]. However,in this reaction, prior to substrate preparation, anhydrous sodiumsulfate were added. The final moisture concentration of the reactionmixture was in the range of 1–2%. Yamane et al. [10] found thattoo high water concentration did not enhance the monoglyceridesconversion; instead, it increased the by-product formation of freefatty acid. The highest yield of monoglycerides was obtained at 10%moisture, which is other than the enzyme prepared from CALB.
Author, Piyatheerawong et al. [12] mentioned that commer-cially available immobilized enzymes prepared from candidaAntarctica lipase B, exhibited highest product formation at very lowmoisture content (less than 1 wt%). Due to the hydrophilic behaviorof both glycerol and water, it is very difficult to separate glycerolless than 1% of moisture content. Thus, the systems have to be inthe range of 1–2% of moisture content. In this condition, it wasfound that Fermase 10000 is very active and product conversion ismaximum.
4. Optimized condition for glycerolysis reaction
The optimum conditions for monoglycerides production is 15%of enzyme with respect to oil, oil to glycerol 1:5 molar ratio, co-
or conversion of sunflower oil to food based emulsifiers, Catal. Today
solvent was t-butanol in 1:50 molar ratio. The economical timeperiod for the optimized condition is 5 h at 200 rpm in shakingincubator at 50 ◦C. In the above-defined condition, the yield ofmonoglycerides conversion is in the range of 75–79%.
Fig. 6. Comparison of %MG conversion with Fermase 10000 and Novozym.
. Compare between Fermase 10000 with Novozyme 435
The optimized condition was compared with Novozyme 435.he comparative study for both the conversions is presented inig. 6. It is observed the reaction condition; the conversion of mono-lycerides is almost similar in both the enzymes after 5 h of reactionime.
However, in consideration with economic point of view,ovozyme 435 is more than five times expensive than Fermase0000. The availability is also limited in compared with Fermase0000. So concern with availability and economical problems, itas suggested Fermase 10000 is a suitable enzyme for conversion
f triglycerides to monoglycerides from Sunflower oil.
. Reusability of enzyme and other reactant
The recovered enzyme from the glycerolysis reaction was re-sed at the above-optimized condition after proper washing and
Please cite this article in press as: M.K. Naik, et al., Enzymatic glycerolysis f(2013), http://dx.doi.org/10.1016/j.cattod.2013.11.005
ry. The activity was found to be reducing after each run. In therst run, the conversion was 75–80% followed by 40–50% in theecond run and less than 25% in the third run. However, extensivetudy needed to check the activity of the enzyme.
[[
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PRESSay xxx (2013) xxx– xxx 5
After the reaction, the reaction product was settled in a sepa-rating funnel and excess glycerol was recovered from the bottomlayer. The extra co-solvent (∼20–30%) present in the reaction mix-ture was recovered by the rotary evaporator, which will be helpfulfor cost evaluation.
7. Conclusion
The glycerolysis reaction for conversion of monoglycerides fromsunflower oil using 10000 is very efficient and economical whereconversion is 75–80%. The same t-butanol is best-suited co-solventfor this reaction compared with other organic solvents. The oil toglycerol molar ratio was suited at 1:5 and oil to co-solvent was 1:50.The optimum reaction period at mild temperatures (50 ◦C) and lowenzyme concentrations (∼15 wt%) in short reaction times (5 h). Ingeneral, this optimum process as well as Fermase 10000 will bevery helpful to carry out the reaction in commercial level.
Acknowledgements
Financial support for the project (DST/TSG/AF/2011/47-G) fromfunding agency Department of Science and Technology (DST), NewDelhi is gratefully acknowledged.
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