Flavour ester, a short-chain ester is the category of compound that widely distributed in nature. This sort of ester besides known as a carboxylic acid ester is extensively used in nutrients, drinks, cosmetics and pharmaceutical industries. Flavour esters are all right organic compound that organizing portion of natural olfactory properties in flowers and fruit. Traditionally, this sort of compounds has been isolated from natural beginnings such as flowers, fruits and veggies. However, these natural spirit esters extracted from works stuffs are frequently limited and expensive for commercial usage. Therefore, it is economically of import to synthesise flavour ester utilizing cheaper and more loosely available stuff to run into the consumer demand.

Conventional chemical synthesis of flavour ester between acid and intoxicant utilizing fouling liquid acids as a accelerator, necessitate post-treatment. Therefore, the usage of biotechnology appears to be an attractive in assorted ester readyings under milder conditions and the merchandise may be given the natural label. Therefore, the usage of enzymes, such as lipases as biocatalysts may offer many important advantages over chemical synthesis such as lower energy demand since enzymes function under mild reaction conditions and enhanced selectivity and quality of merchandise.

However, the usage of native lipase signifier brings about important practical jobs such as the high cost of the enzyme. As a mean of cut downing the cost, the usage of immobilized lipases is significantly advantageous. Immobilized lipase are lipases affiliated to solid stuffs, which make them easy be recovered from the reaction mixtures, therefore offers reutilizations of the biocatalyst and thereby doing the procedure economically executable. Another purpose of utilizing immobilized lipase is that the functional activities every bit good as the stableness of enzymes are improved compared to the native lipase.

Optimization of reaction procedure is really of import in enzymatic synthesis in order to better the reaction public presentation. Since ester synthesis is a specific job as it is affected by assorted factors depending upon the reaction status used. The conventional method of optimisation procedure involves changing one parametric quantity at-a-time and maintaining the other invariable. Bing single-dimensional, this method is inefficient as it fails to understand relationships between the variables ( reaction clip, temperature, sum of enzyme and substrate concentration ) and response ( per centum transition ) . These processs are clip consuming, burdensome, require a batch of experimental informations sets and do non supply information about the common interactions of the parametric quantities and sometimes it besides can take to misunderstanding of the consequences. Therefore understanding and mold of both conventional and synergistic effects of of import parametric quantities are indispensable in order to obtain a high public presentation synthesis.

A statistical based technique normally used for this intent is Response Surface Methodology ( RSM ) . This method which is an efficient statistical tool for optimisation of multiple variables with several designs is an effectual technique for anticipation and the probe of complex procedures. By transporting out merely a few selected experiments such designs explain the reaction wholly conveying out the finer inside informations. Statistical analysis of Response surface methodological analysis quantifies the relationships between variables and response ( output ) therefore determines the optimal operating conditions in a procedure. This methodological analysis leads to antic discoveries in procedure apprehension, therefore bettering quality, cut downing costs and increasing net incomes.

Therefore, the intent of this research is to analyze the optimisation procedure of green synthesis of spirit esters via lipase-catalyzed reactions in solvent free system via statistical attack of Response surface methodological analysis ( RSM ) . Nonyl caprylate which is known as the chief constituent in citrous fruit and rose spirit was the targeted spirit ester in this probe. It is necessary to place the of import factors that affect the public presentation of the procedure in little graduated table system, so that a suited attack of scaling-up and a design theoretical account could be proposed prior to commercialisation.

## Materials and Methods

## Materials

Novozym 435 as 10,000 PLU ( from Candida Antarctica lipase immobilized onto macroporous acrylin rosin ) was received from Novo Nordisk ( Denmark ) . Nonanol ( pureness, 98 % ) and caprylic acid ( pureness, 97 % ) were obtained from Merck ( Germany ) . All other reagents were of analytical class and used as received.

## Enzymatic Synthesis

The reaction system consisted of nonanol and caprylic acid ( 1:1 ) and 5 % of enzyme ( w/w ) were mix in screw-capped phial. The mixture was incubated at 37 & A ; deg ; C utilizing a horizontal waterbath shaker. The agitating velocity was set at 150 revolutions per minute and the reaction mixture was continuously reacted for 12 hours.

## Analysis of Reaction Product

Determination of the per centum transition of ethyl valerate ( % ) :

The per centum transition ( % ) of nonyl caprylate was measured by finding the staying unreacted fatty acids in the reaction mixture by titration with 1.0 M NaOH in an automatic titrator ( Methrom, Switzerland ) . All the samples were assayed in triplicate and the experiment was repeated twice.

Conversion of flavour ester ( % ) =

Volume of NaOH used ( without enzyme ) – Volume of NaOH used ( with enzyme ) X 100 Ex. ( 1 )

Volume of NaOH used ( without enzyme )

## Experimental Design

A five-level, four-factor cardinal composite rotatable design ( CCRD ) was employed, necessitating 30 experiments. The fractional factorial design consisted of sixtheen factorial points, eigth axial points and six Centre points. The variables and their several degrees are presented in Table 1. Table 2 represents the existent experiments carried out for developing the theoretical account. The informations obtained were fitted to a second-order multinomial equation:

Ex. ( 2 )

Where Y= % transition of flavour ester, b0, Bi, bii and bij are changeless coefficients and elevens are the uncoded independent variables. Subsequent arrested development analysis, analysis of discrepancy ( ANOVA ) and response surfaces were performed utilizing Design Expert Software ( version 7.1.6 ) from stat easiness ( Minneapolis, MN ) . Optimum reaction parametric quantities for maximal transition were generated utilizing the package ‘s numerical optimisation map.

## Table 1. Coded and existent degrees of variables for design of experimenta

Coded values of variables

Factor

Name

Unit of measurement

-?

-1

0

1

?

A

Time

hr

0.5

3

5

8

10.5

Bacillus

Enzyme Amount

% ( w/w )

5

10

15

20

25

C

Temperature

& A ; deg ; C

20

30

40

50

60

Calciferol

Shaking Speed

revolutions per minute

50

100

150

200

250

aStudy type, response surface ; No. of experiments, 30 ; design, CCRD ; response, Y1, name, flavour ester, unit, % transition.

## Table 2. Design matric of the existent experiments carried out for developing the theoretical account

## Standard

## A ( hr )

## B % ( w/w )

## C ( & A ; deg ; C )

## D ( revolutions per minute )

## Actual ( % )

## Predicted ( % )

## 1

## 3

## 10

## 30

## 100

## 81.337

## 82.116

## 2

## 8

## 10

## 30

## 100

## 85.722

## 85.976

## 3

## 3

## 20

## 30

## 100

## 89.603

## 89.114

## 4

## 8

## 20

## 30

## 100

## 90.361

## 91.112

## 5

## 3

## 10

## 50

## 100

## 84.115

## 83.777

## 6

## 8

## 10

## 50

## 100

## 85.785

## 86.013

## 7

## 3

## 20

## 50

## 100

## 89.783

## 89.731

## 8

## 8

## 20

## 50

## 100

## 89.898

## 90.154

## 9

## 3

## 10

## 30

## 200

## 86.937

## 86.510

## 10

## 8

## 10

## 30

## 200

## 88.716

## 89.119

## 11

## 3

## 20

## 30

## 200

## 89.687

## 89.810

## 12

## 8

## 20

## 30

## 200

## 90.441

## 90.607

## 13

## 3

## 10

## 50

## 200

## 87.210

## 86.811

## 14

## 8

## 10

## 50

## 200

## 87.528

## 87.846

## 15

## 3

## 20

## 50

## 200

## 89.543

## 89.117

## 16

## 8

## 20

## 50

## 200

## 88.817

## 88.339

## 17

## 0.5

## 15

## 40

## 150

## 83.149

## 83.829

## 18

## 10.5

## 15

## 40

## 150

## 87.720

## 86.861

## 19

## 5.5

## 5

## 40

## 150

## 84.756

## 84.412

## 20

## 5.5

## 25

## 40

## 150

## 91.688

## 89.069

## 21

## 5.5

## 15

## 20

## 150

## 90.463

## 89.748

## 22

## 5.5

## 15

## 60

## 150

## 88.556

## 89.092

## 23

## 5.5

## 15

## 40

## 50

## 88.019

## 87.389

## 24

## 5.5

## 15

## 40

## 250

## 89.467

## 89.918

## 25

## 5.5

## 15

## 40

## 150

## 88.698

## 89.069

## 26

## 5.5

## 15

## 40

## 150

## 89.657

## 89.069

## 27

## 5.5

## 15

## 40

## 150

## 88.388

## 89.069

## 28

## 5.5

## 15

## 40

## 150

## 89.207

## 89.069

## 29

## 5.5

## 15

## 40

## 150

## 88.966

## 89.069

## 30

## 5.5

## 15

## 40

## 150

## 89.499

## 89.069

## Result and Discussion

## 3.1 Model adjustment and ANOVA

The coefficients of the empirical theoretical account and their statistical analysis, evaluated utilizing Design Expert Software, are presented in Tables 3-5. The exemplary F-value of 19.17 with a ‘Prob & A ; gt ; F ‘ value of 0.0001 implied that the theoretical account was important at the 1 % assurance degree. The high coefficient of finding ( R2= 0.9471 ) of the theoretical account indicated the suitableness of the theoretical account for adequately stand foring the existent relationship among the parametric quantities studied. A high value of R2 ( & A ; gt ; 0.950 ) has been besides reported by Hari Krishna et Al [ 6 ] , for the lipase-catalysed synthesis of isoamyl isobutyrate and by Jei et al [ 7 ] for the enzymatic optimisation of propene ethanediol monolaurate by direct esterification. In this survey, quadratic theoretical account was shown to be the most important theoretical account due to the low value of chance ( P=0.0001 ) and high value of coefficient finding ( R2=0.9471 ) . Similar quadratic response theoretical accounts have been reported by Shieh et al [ 8 ] and Chen et al [ 9 ] in the optimisation of lipase-catalyzed synthesis of biodiesel ( soybean oil methyl ester ) and kojic acerb monolaurate, severally. The theoretical account indicates the important footings was observed for linear ( A and C ) , quadratic ( C ) and synergistic consequence ( AC ) harmonizing to the value of ‘Prob & A ; gt ; F ‘ & As ; lt ; 0.050. The concluding equation was derived in footings of coded factors for the synthesis of ethyl valerate as shown in Equation ( 3 ) :

Y = +77.01 + 3.23A + 12.10B + 0.40C + 1.36D – 3.51AB – 1.32AC + 0.63AD – 0.11BC – 1.44BD + 1.25CD – 0.89A2- 4.83B2- 0.57C2- 0.18D2

Ex. ( 3 )

where A is the clip ; B is the temperature ; C is the sum of enzyme ; D is the agitating velocity.

## Table 3. Statistical analysis: Analysis of variance

Beginning

Sum of squares

Degrees of freedom

Mean Square

F-value

Prob & A ; gt ; F

Model

155.92

14

11.14

24.74

& A ; lt ; 0.0001

Residual

6.75

15

0.45

Lack of tantrum

5.59

10

0.56

2.41

0.1720

Pure mistake

1.16

5

0.23

Entire

162.67

29

aSignificance at ‘Prob & A ; gt ; F ‘ is & A ; lt ; 0.0500

## Table 4. Statistical analysis: arrested development analysis

Std. Dev.

0.67

Mean

88.12

R-squared

0.9585

Adj-R-Squared

0.9198

Pred-R-Squared

0.7917

Adeq Precision

20.419

## Table 5. Statistical analysis: coefficient of theoretical accounts

Factor

Coefficient Estimate

Prob & A ; gt ; F

Intercept

89.07

& A ; lt ; 0.0001

A-Time

0.76

& A ; lt ; 0.0001

B-Temperature

1.86

& A ; lt ; 0.0001

C-Enzyme Amount

-0.16

0.2496

D-Shaking Speed

0.63

0.0003

AB

-0.45

0.0164

Actinium

-0.39

0.0331

Ad

-0.30

0.0936

BC

-0.25

0.1590

Bachelor of divinity

-0.91

& A ; lt ; 0.0001

Cadmium

-0.33

0.0698

A2

-0.93

& A ; lt ; 0.0001

B2

-0.23

0.0875

C2

0.088

0.5042

D2

-0.10

0.4298

aA = Time ; B = Temperature ; C = Enzyme sum ; D = Shaking Speed.

bSignificance at ‘Prob & A ; gt ; F ‘ is & A ; lt ; 0.0500