(560p) Ultrasound-Assisted Production of Ethyl Butyrate VIA a Lipase Cocktail

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ULTRASOUND-ASSISTED PRODUCTION OF ETHYL BUTYRATE VIA A

LIPASE COCKTAIL

Fernando C. de Sousa Filho

1

, Roberto M. F. de Freitas

1

, Bárbara K. N. Gadelha

1

,

Ellefson E. S. de Oliveira

1

, Rodolpho R. C. Monteiro

2

, Paula J. M. Lima

2

, Maria C. M

de Souza

1

and José C. S dos Santos

1,*

.

1

Instituto de Engenharias e Desenvolvimento Sustentável, Universidade da Integração

Internacional da Lusofonia Afro-Brasileira, CEP 62790-970, Redenção, CE, Brazil.

2

Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do

Pici, Bloco 709, CEP 60455-760 Fortaleza, CE, Brazil.

*jcs@unilab.edu.br

1. INTRODUCTION

Enzymes are highly specific and efficient biocatalysts that catalyze various

chemical and biochemical reactions under certain conditions of temperature, pH,

reaction medium, etc (BILAL, et al., 2018). Lipases are enzymes used in biocatalysis,

as they have a broad range of compatibility with several substrate species and high

stability under multiple conditions. In addition, lipase properties, including selectivity,

specificity and activity are easily modulated (SANTOS et al., 2015). The concept of

combi-lipases have an increasing interest for heterogeneous substrates, such as oils,

once the use of an individual lipase may hardly be “optimal” for all likely components

of the oils (ORTIZ et al., 2019).

Ultrasound is the sound energy at frequencies above the range that is audible to

humans (> 20 kHz) and which needs a medium to propagate. The positive effects of this

technology on enzyme activity and can its potential to accelerate enzymatic reactions,

show great potential for industrial applications. However, high frequencies and

potencies can cause disruption of the structure of the enzyme, causing denaturation of

this macromolecule, while low frequencies and potencies will not result in the desirable

effect of cavitation (BODESODE & RATHOD, 2014).

Flavors are composed of different organic chemicals, such as hydrocarbons,

alcohols, aldehydes, ketones, acids, esters or lactones. The low volatility and low

molecular weight are responsible for a series of sensorial sensations. Flavor esters are

widely found in nature and confer pleasant attributes with fruity, floral, spicy and

creamy aromas. These properties enable a wide variety of food applications in many

beverages, sweets, jellies, compounds, wines and dairy products.

Ethyl butyrate is an flavor ester obtained by esterifying a fatty acid or

monocarboxylic acid (butyric acid) with an alcohol (ethyl alcohol). This ester is an

important component of aromas of many fruits, for example: pineapple, passion fruit,

strawberry and apple (GUMEL; ANNUAR, 2016).

Hereupon, this communication has as main objective to determine the best

cocktail of Lipase B from Candida antarctica (CALB), Thermomyces lanuginosus

(TLL) and Rhizomucor miehei (RML) for the ultrasound-assisted production of ethyl

butyrate.

2. MATERIALS

Lipases from Thermomyces lanuginosus (immobilized on a silicate support,

Lipozyme TL-IM), Rhizomucor miehei (immobilized on an anion-exchange resin,

Lipozyme RM-IM) and lipase B from Candida antarctica (immobilized on a

macroporous resin, Novozym 435) were kindly donated by Novozymes (Madrid,

Spain). All other chemicals were of analytical grade and they have been used without

any further purification.

The equipment used in all experiments was an ultrasonic bath (Unique Inc.,

model USC 2800A, Brazil). The equipment presents the capacity volume of 9.5 L with

the following dimensions: 300 × 240 × 150 mm (length × width × height). Two disc

transducers were placed at the bottom of the reactor. The ultrasonic frequency was 37

kHz and the total ultrasonic power 300 W. Additionally, the equipment has temperature

control.

3. METHODOLOGY

In order to determine the best combination of lipases (TLL, RML and CALB)

for the production of ethyl butyrate, a 3-factor mixture design and triangular surface

analysis was performed using Statistica® 10 (Statsoft, USA). For the esterification, it

was considered a 1:1 molar ratio (butyric acid/ethyl alcohol) and a biocatalyst mass of

10% of the butyric acid mass. The reactions were conducted at ultrasonic radiation at 37

kHz and 300 W for 3 hours and 35 °C. The reactions were performed in triplicate and

the results expressed as means and standard deviations. Conversion was monitored by

determining the acid index through the Ca 5-40 AOCS method.

4. RESULTS AND DISCUSSIONS

The combinations of lipases (CAL-B, RML and TLL) resulting from the

simplex-centroid experimental design with interior points and centroid and their

respective conversion rates are presented in Table 1. As can be seen in Table 1, the best

cocktail of lipases for the ultrasound-assisted production of ethyl butyrate was that

proposed by run number 10, which has the same amount of each lipase in study,

achieving a conversion of 80.41%.

Table 1 – Experiments performed in the mixture design. Reaction medium: 1:1 molar

ratio (butyric acid/ethyl alcohol), biocatalyst mass of 10% of the butyric acid mass. The

reactions were conducted at ultrasonic radiation at 37 kHz and 300 W for 3 hours and

35 °C. Further information are given on Section 3.

RUN

CALB

RML

TLL

CONVERSION (%)

1

1.000

0.000

0.000

76.81 ± 2.32

2

0.000

1.000

0.000

35.80 ± 4.14

3

0.000

0.000

1.000

46.55 ± 1.56

4

0.500

0.500

0.000

77.76 ± 2.57

5

0.500

0.000

0.500

38.96 ± 1.83

6

0.000

0.500

0.500

37.84 ± 3.66

7

0.667

0.167

0.167

79.25 ± 0.89

8

0.167

0.667

0.167

46.97 ± 3.81

9

0.167

0.167

0.667

37.74 ± 2.45

10

0.333

0.333

0.333

80.41 ± 0.94

Analyzing the behavior of each lipase in individual, CAL-B (76.81%) has

presented the best conversion when compared to TLL (46.55%) and RML (35.80%),

respectively. Also, for the combinations of only two lipases, CALB-RML and CALB-

TLL have shown the better values than that for RML-TLL (37.84%). By these results

and by analyzing the Pareto chart (Figure 1), for the ultrasound assisted production of

ethyl butyrate, it can be stated that CALB, among the lipases studied, has the major

effect on the conversion. TLL and RML are both 1,3-regio specific lipases, whereas

CALB is a non-specific lipase. The combination of the 1,3-specific with non-specific

lipases may have increased the reaction rate by attacking the different parts of the

substrates, showing the specificity of RML and TLL for the substrate and the high

activity of CALB, even being a non-specific lipase.

Figure 1 – Pareto chart. Reaction medium: 1:1 molar ratio (butyric acid/ethyl alcohol),

biocatalyst mass of 10% of the butyric acid mass. The reactions were conducted at

ultrasonic radiation at 37 kHz and 300 W for 3 hours and 35 °C. Further information are

given on Section 3.

Besides, the results shown above point out the efficiency of the ultrasonic

technology for the production of ethyl butyrate when compared to traditional

techniques. Souza et al., (2017), using CALB obtained conversions higher than 90% for

the production of ethyl butyrate under 25 °C, 150 rpm and a molar ratio of 1:1 after 8

hours of reaction, while in this study conversions of around 80% were obtained after

only 3 hours of reaction. Badgujar and Bhanage (2015) produced benzyl butyrate, anisil

butyrate and o-cresyl butyrate using the ultrasound technique and obtained a conversion

yield of 99% after 3 hours of reaction at 52 °C, while in this study conversions of

around 80% were obtained under 35 °C.

5. CONCLUSIONS

Here, the use of a cocktail of lipases for the assisted-production of ethyl butyrate

was proposed. The optimum combination was found to be 33,3% of RML, 33,33% of

TLL and 33% of CALB, showing the specificity of RML and TLL for the substrate and

the high activity of CALB, even being a non-specific lipase. Besides, the results shown

above point out the efficiency of the ultrasonic technology for the production of ethyl

butyrate when compared to traditional techniques.

REFERENCES

AOCS – American Oil Chemists Society; Official and Tentative Methods, 3ª ed.,

Chicago, 1985, vol.

BADGUJAR, K.C.; BHANAGE, B.M. The combine use of ultrasound and lipase

immobilized on co-polymer matrix for efficient biocatalytic application studies.

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http://dx.doi.org/10.1016/j.molcatb.2015.09.012.

BANSODE, S.R.; RATHOD, V.K. Ultrasound assisted lipase catalysed synthesis of

isoamyl butyrate. Process Biochemistry, v. 49, n. 8, p.1297-1303, 2014. Elsevier BV.

http://dx.doi.org/10.1016/j.procbio.2014.04.018.

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120, p.2530-2544, 2018. Elsevier BV. http://dx.doi.org/10.1016/j.ijbiomac.2018.09.025.

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6, n. 1, p.6-24, 11 jan. 2016. Springer Nature. http://dx.doi.org/10.1007/s13205-015-

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ORTIZ, C. et al. Novozym 435: THE “PERFECT” LIPASE IMMOBILIZED

BIOCATALYST?. Catalysis Science & Technology, p.1-126, 2019. Royal Society of

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SANTOS, J.C.S. dos et al. Versatility of divinylsulfone supports permits the tuning of

CALB properties during its immobilization. Rsc Advances, v. 5, n. 45, p.35801-35810,

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SOUZA, M.C.M. de et al. Production of flavor esters catalyzed by lipase B from Candida

antarctica immobilized on magnetic nanoparticles. Brazilian Journal of Chemical

Engineering, v. 34, n. 3, p.681-690, 2017. FapUNIFESP (SciELO).

http://dx.doi.org/10.1590/0104-6632.20170343s20150575.