Lactonization Castor Oil (Ricinus Communis) using Lipase B from

Candida Antarctica Recombined Aspergillus oryzae as Bioflavor

Galuh Alya Stywarni

, Elvina Dhiaul Iftitah

and Arie Srihardyastutie

Department of Chemistry, Faculty of Mathematics and Science, Brawijaya

University, Malang, Indonesia

Keywords: Castor Oil (Ricinus communis), Lactonization, Lipase, Bioflavor.

Abstract: Lactone is a widely flavor that is used in food production. Lactonization using microbial or enzyme has natural

labelled products, has a higher economic value than artificial products and is safe for the environment.

Lactonization of castor oil (Ricinus communis) using lipase B from Candida antarctica recombined

Aspergillus oryzae (T = room, 40ºC) for 24, 48 and 72 h were investigated. The lactonization reaction was

carried out using a magnetic hotplate stirrer with the reaction system consisting of castor oil, n-hexane solvent,

Na2CO3 solution, and lipase biocatalyst. Lactonization castor oil products were analysed using GC-MS. At

T = room, the major products were ester: methyl ricinoleate, 53.64% (t = 24 h) and other products were fatty

acids and lactone. Lactone: γ-dodecalactone, 1.75% (t = 48 h) was a minor product. Whereas at T = 40ºC,

only produced ester, the major product was methyl ricinoleate, 81.33% (t = 72 h).

1 INTRODUCTION

One of the potential sources of natural-based raw

materials that are widely used in industry is castor oil.

Castor oil consists of thick yellow liquid, has a

characteristic odour with a molecular weight of

933.45 g/mol, a density of 0.95 g / cm3 and a boiling

point of 313°C (Moradi et al., 2013). The content of

castor oil consists of ricinoleic acid, linoleic acid,

oleic acid, stearic acid, palmitic acid,

dihydroxystearic acid, linoleic acid, and eicosanoic

acid (Farbood and Willis, 1985). The main

component of castor oil is ricinolein, a glyceride

from ricinoleic acid. Ricinoleic acid has three

functional groups namely ester linkage, double

bonds and hydroxyl groups which are used as

sources of renewable raw materials in chemical

reactions, modification, and transformation into

useful products (Wache et al., 2001). In the food

industry, castor oil potential produces bioflavor.

Various lipase-producing microbes have been

reported as catalyse bioflavor (γ-decalactone) using

castor oil substrate or ricinoleic acid (12-hydroxy-9-

octadecenoic acid).

γ-Decalactone as a flavouring agent has fruit,

creamy, peach, apricot, and fatty taste. In enzymatic

biotransformation, the substrate is degraded through

α-oxidation to produce 4-hydroxidecanoic acid, then

cyclization to

γ-decalactone (Gutman et al., 1989)

Based on research by Gotz et al. (2013), immobilized

B lipase from Candida antarctica able to catalyse the

formation of (S) -γ-valerolactone from a substrate

(S) -ethyl-4- hydroxy pentanoate with a yield of

90% (Antczak et al., 1991). According to Gutman

et al. (1989), the lactonization reaction rate affected

by the hydrophobicity of the solvent, n-hexane

solvent is two times faster than ether and four times

faster than chloroform (Khan and Rathod, 2018).

2 MATERIALS AND METHODS

2.1 Chemicals and Enzymes

Candida antarctica lipase B (recombinant from

Aspergillus oryzae) (1800 U/gram), n- hexane, and

sodium carbonate were obtained from Sigma-

Aldrich. Castor oil were obtained from Organic

Supply Co.

2.2 GC-MS Analysis of Castor Oil

(Ricinus communis)

Transesterification reaction castor oil was carried out

Stywarni, G., Iftitah, E. and Srihardyastutie, A.

Lactonization Castor Oil (Ricinus Communis) using Lipase B from Candida Antarctica Recombined Aspergillus oryzae as Bioﬂavor.

DOI: 10.5220/0009955000370040

In Proceedings of the 2nd International Conference of Essential Oils (ICEO 2019), pages 37-40

ISBN: 978-989-758-456-5

to determine the components of fatty acids. 50 g

castor oil, 38 mL of ethanol

and 1 mL of H

1 M

were put in 100 mL

Erlenmeyer flasks. The mixture

was refluxed at 60-70ºC for 2 h. Then, Saturated NaCl

was added to separate organic and water phase. The

organic phase dehydrated by anhydrous

and

dissolved in n-hexane (1:40, v/v).

Gas Chromatography (GC) equipped with a Mass

Spectra (MS) detector and Restrex

Rxi®-1MS

capillary column. Oven temperature

was held at 40 -

250ºC; injection temperature was 250ºC; The carrier

gas, helium, was adjusted to a linear velocity 0.7

mL/min and 24.9 kPa. The injection volume

into the

GC apparatus was 0.5µl.

2.3 Castor Oil Lactonization

The r eact ion was carried out in 100 mL Erlenmeyer

flasks, containing 6 g castor oil,

40 mL of n-hexane

solvent,1 mL of Na

solution, and 0.1 g Candida

antarctica recombined Aspergillus oryzae. The

reactions were stirred using magnetic hotplate stirrer

at room temperature and 40ºC for 24, 48 and 72 h.

Then the pH of the mixture was measured. Each

sample was centrifuged to separate the enzyme a n d

the oil phase. The samples dissolved in n-

hexane (1:20, v/v).

Gas Chromatography (GC) equipped with

a Mass Spectra (MS) detector and Restrex Rtx®-

5MS capillary column. Oven temperature w a s

held at 40 - 250ºC; injection

temperature

was

250ºC;

The

carrier

gas,

helium, was adjusted

to a linear velocity 1.01 ml/min and 50 kPa. The

injection samples into the GC apparatus was 0.5µl.

3 RESULTS AND DISCUSSION

3.1 Analysis of Castor Oil

Substrate (Ricinus communis)

Generally, the composition of ricinoleic acid in castor

oil comprises approximately 90%, while the

composition of other fatty acids: linoleic acid, oleic

acid, stearic acid, palmitic acid, dihydroxystearic

acid, linolenic acid, and eicosanoic acid less than

5% (Kourist and Hilterhaus, 2015). Based on

analysis using GC-MS, the highest % concentration

transesterification product castor oil was methyl

ricinoleate (88.666%) (Table 1). So that, ricinoleic

acid is the major component of castor oil. Some fatty

acid components were not found in castor oil

substrate, but % concentration of ricinoleic acid as

the major component was good quantity.

Table 1: Trans-esterification product of castor oil.

Cons.

Compound

0.872

Methyl-14-methyl

pentadecanoate

4.285 Methyl-9-12-

octadecadienoate

4.547 Methyl-11-

octadecenoate

1.630 Methyl octadecanoate

88.666 Methyl ricinoleate

2.4 Effect of Temperature and

Reaction Time

Lactonization using Candida antarctica lipase B

recombined Aspergillus oryzae not only produce

lactone. The lactone was γ- dodecalactone only

formed at room temperature for 48 h. (Table 2).

Table 2: Effect temperature and reaction time on lactone

formation.

Temperature

(ºC)

Reaction

Time (h)

Lactone

room

24 -

√

72 -

24 -

48 -

72 -

Biotransformation product of castor oil at room

temperature were esters, fatty acids and lactone.

Whereas at 40ºC only formed esters (Table 3).

Table 3: Biotransformation product of castor oil.

(ºC)

(h)

Compound % Area

Ambient

24 Methyl ricinoleate 53.64

9-octadecenoic acid 4.37

γ-Dodecalactone 1.75

Methyl dodecanoate 4.58

Dodecanoic acid 1.53

Methyl-9-

octadecenoat

1.59

Methyl dodecanoat 1.61

Methyl ricinoleat 12.77

40 24 Methyl ricinoleat 69.90

48 Methyl ricinoleat 64.95

72 Methil ricinoleat 81.33

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

Figure 1. Estimated mechanism formation of fatty acid and

ester. Condition of reaction: (a)T= room, t = 24 h; (b)T=

room, t = 48 h; (c)T= room, T = 72 h; (d)T= 40ºC, t =24

h; (e)T= 40ºC, t = 48 h; (f) T = 40ºC, t = 72 h.

As the date in Table 3 show, Biotransformation of

castor oil at room temperature and 0ºC produces

esters (methyl ricinoleate) as the major product. It can

be assumed that before the esterification reaction,

triglyceride was hydrolysed to fatty acids, ricinoleic

acid (undetectable). The optimal yield of methyl

ricinoleate at 40ºC for 72 h (81.33%), is higher than

the methyl ricinoleate at room temperature.

Formation C

Fatty acid products at

room

temperature (9-octadecenoic acid, t =

48 h) and C

(

dodecanoic acid, t = 48 h)

showed that lipase B

Candida antarctica recombined Aspergillus oryzae

had ability to hydrolysis triglyceride and shortening

fatty acid carbon chain. This is probably hydrolysis

reaction because lipase had active side catalytic:

serine, histidine, and aspartate (Veld, 2010). The

shortening C

fatty acid chain to C

can be

assumed

at beta carbon position that occur oxidation into

carbonyl groups as much as three times. Source

water in hydrolysis

reaction form added Na

solution

component reaction. Then, fatty acid was

probably forming ester. The fatty acid is then

probably transformed into ester. Formation reaction

of fatty acid and ester showed at Figure 1 9-

octadecanoic acid probably to form

methyl-9-

octadecanoic

(t = 48 h), dodecanoic acid probably to

form methyl dodecanoate (t = 48 and 72 h). Methyl

dodecanoate product (t = 72 h, 1.61%), lower than

methyl dodecanoate (t = 48 h, 4.58%).

Figure 2. Estimated mechanism formation of

gamma-dodecalactone. Reaction: (a) hydrolysis,

(b) hydroxylation (c) shortening of carbon chains

(d) lactonization.

Lactonization reaction to form γ- dodecalactone

is probably from 9- octadecanoic acid, then

hydroxylated to form 10-hydroxy octadecanoic acid

(undetectable), after that undergoes a carbon chain

Lactonization Castor Oil (Ricinus Communis) using Lipase B from Candida Antarctica Recombined Aspergillus oryzae as Bioﬂavor

shortening to form 4-hydroxy dodecanoic acid

(undetectable), and it was occurring lactonization

become γ-dodecalactone. Formation of γ-

dodecalactone product is also probably from

dodecanoic acid (Figure 2). The mechanism of γ-

dodecalactone formation (Figure 2) refers to Han et

al. (1995) who use Mortierella isabellina on

dodecanoic acid substrate and Haffner et al. (1996)

using the sporobolomyces odour on 9-octadecanoic

acid (oleic acid) that occur hydroxylation

(Goswami et al., 2013) to form γ-dodecalactone

The target compound that is γ-decalactone was

not formed in

biotransformation

of castor oil at room

temperature and 40ºC, it is estimated that due to

ideal biotransformation reaction conditions for

esterification, so that the hydroxylation reaction to

form ricinoleic acid (not detected) as substrate

probably convert quickly to ester (methyl

ricinoleate). The formation of another lactone (γ-

dodecalactone, 1.75%) as minor product at room

temperature for 48 h probably so because the

hydrolysis of 9-octadecenoic acid and dodecanoic

acid was formed during the 48 h, so that

lactonization (intra-esterification reaction) is

possible at these time.

4 CONCLUSIONS

Lactonization of castor oil only produces lactone at

room

temperature

for 48 h. The lactone product was γ-

dodekalakton as minor product (1.75%). The major

products biotransformation was methyl ricinoleat

(T=room, t=24 h: 53.64%); (T=room, t= 72 jam:

12.77%); (T=40ºC, t=24 h: 69.90%); (T=40ºC, t=48 h:

64.95%); (T=40ºC, t=72 h 81.33%).

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ICEO 2019 - 2nd International Conference of Essential Oil Indonesia