**3. Results and discussion**

#### **3.1. Food-grade enzyme preparation activity**

In order to achieve the optimum cellulolytic activity of the enzyme preparation, filter paper units (FPU. mL−<sup>1</sup> ) of each one were first determined. Crystalzyme PML-MX and Cellulase 17600 L showed the highest cellulolytic activities with 6.96 and 5.60 FPU. mL−<sup>1</sup> , respectively. This result is probably due to the presence of cellulases in these enzymatic preparations, since the determination of the activity is carried out with filter paper, which is formed by cellulose [42], while the enzymatic preparations, Crystalzyme 100XL and Crystalzyme Cran, showed lower activities with 2.65 and 2.38 FPU mL−<sup>1</sup> , respectively, and do not contain cellulases (**Table 1**). These results were used as reference to define the amount of enzyme needed to hydrolyze the neem seed cellulolytic structures and extract the highest concentration of azadirachtin.

#### **3.2. pH and temperature determinations**

Determination of the optimal reaction conditions was carried out at 50°C, taking into account that temperature is indicated by the enzyme preparation supplier as the optimum. However, in the evaluations, dehydrated neem seed was used instead of filter paper or crystalline cellulose. **Figure 1** shows the determinations of the optimum pH of each enzyme preparations, and the results indicate that all enzyme preparations perform the hydrolysis of the neem seed under very similar pH conditions regardless of the combinations of cellulolytic activity they contain. Optimal pH of the enzymatic preparations was 5.0 for Crystalzyme Cran and 4.5 for Crystalzyme PML-MX, Cellulase and Crystalzyme 100XL. Of the four enzyme preparations, Crystalzyme PML-MX exhibited the highest hydrolysis of the neem seed with 2.2114 (± 0.1879) mg reducing sugars mL−<sup>1</sup>


**Figure 1.** Optimum pH of food-grade enzyme preparations: (A) Crystalzyme PML-MX, (B) Cellulase 17600, (C) Crystalzyme

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Cran, and (D) Crystalzyme 100XL.

**Table 1.** Enzymatic activities of the food-grade enzyme preparations.

Biopesticide of Neem Obtained by Enzyme-Assisted Extraction: An Alternative to Improve the Pest Control http://dx.doi.org/10.5772/intechopen.80028 115

were used to evaluate the differences between means with the statistical program Minitab 17.3. The kinetics of release of reducing sugars and azadirachtin were analyzed by a first-

release rate constant (*k*) were determined, and the results were analyzed by ANOVA (p < 0.05)

In order to achieve the optimum cellulolytic activity of the enzyme preparation, filter paper

result is probably due to the presence of cellulases in these enzymatic preparations, since the determination of the activity is carried out with filter paper, which is formed by cellulose [42], while the enzymatic preparations, Crystalzyme 100XL and Crystalzyme Cran, showed lower

These results were used as reference to define the amount of enzyme needed to hydrolyze the

Determination of the optimal reaction conditions was carried out at 50°C, taking into account that temperature is indicated by the enzyme preparation supplier as the optimum. However, in the evaluations, dehydrated neem seed was used instead of filter paper or crystalline cellulose. **Figure 1** shows the determinations of the optimum pH of each enzyme preparations, and the results indicate that all enzyme preparations perform the hydrolysis of the neem seed under very similar pH conditions regardless of the combinations of cellulolytic activity they contain. Optimal pH of the enzymatic preparations was 5.0 for Crystalzyme Cran and 4.5 for Crystalzyme PML-MX, Cellulase and Crystalzyme 100XL. Of the four enzyme preparations, Crystalzyme PML-MX exhibited the highest hydrolysis of the neem seed with 2.2114 (± 0.1879) mg reducing sugars mL−<sup>1</sup>

**Enzyme preparations Cellulolytic activity Activity (FPU mL−<sup>1</sup>**

Crystalzyme PML-MX Pectinase, endoglucanase, exoglucanase, hemicellulase 6.96 Crystalzyme Cran Pectinase 2.38 Crystalzyme 100XL Pectinase and arabinase 2.65

Cellulase 17600 Endoglucanase, exoglucanase, β-glucosidase, pectinase and

arabinoxylanase

**Table 1.** Enzymatic activities of the food-grade enzyme preparations.

neem seed cellulolytic structures and extract the highest concentration of azadirachtin.

17600 L showed the highest cellulolytic activities with 6.96 and 5.60 FPU.

) of each one were first determined. Crystalzyme PML-MX and Cellulase

mL−<sup>1</sup>

, respectively, and do not contain cellulases (**Table 1**).

, respectively. This

**)**

5.6

(1−e−kt), in which the equilibrium concentrations (Ye

) and its

order empirical equation Yi = Ye

**3. Results and discussion**

mL−<sup>1</sup>

units (FPU.

to determine differences between treatments.

114 Soil Contamination and Alternatives for Sustainable Development

**3.1. Food-grade enzyme preparation activity**

activities with 2.65 and 2.38 FPU mL−<sup>1</sup>

**3.2. pH and temperature determinations**

**Figure 1.** Optimum pH of food-grade enzyme preparations: (A) Crystalzyme PML-MX, (B) Cellulase 17600, (C) Crystalzyme Cran, and (D) Crystalzyme 100XL.

extract. A second reaction was carried out adjusting each hydrolysis to the optimum pH of each enzyme, and the effect of the temperature in the range of 25–70°C was evaluated (**Figure 2**).

Cellulase 17600L, Crystalzyme Cran, and Crystalzyme 100XL shown higher activity at 50°C, while to Crystalzyme PML-MX was at 45°C. These conditions are within the limit considered by [34] to maintain the neem extracts without the loss of azadirachtin. However, it is necessary to evaluate these conditions on the release of azadirachtin, since it is unknown whether the highest hydrolysis of the neem seed ensures maximum concentration of azadirachtin.

#### **3.3. Enzyme concentration**

Results of the enzyme concentration effect over hydrolysis of neem seeds could be observed in **Figure 3**. Reducing sugar release was used as indirect measure of neem seed hydrolysis.

Reactions catalyzed by Crystalzyme PML-MX and Cellulase 17600 L presented the highest hydrolysis of the neem seed, probably due to the presence of cellulases in its composition. A total of 1.8072 (± 0.0021), 2.0635 (± 0.0689), 2.0493 (± 0.0521), and 2.0742 (± 0.0283) g L−<sup>1</sup> reducing sugars were released when 0.5 (3.48 FPU), 1 (6.96 FPU), 2 (13.92 FPU), and 4 mL (27.84 FPU) of Crystalzyme PML-MX were used. On another hand, 1.8034 (± 0.0387), 2.0809 (± 0.0023), 1.9921 (± 0.0456), and 1.9383 (± 0.0781) g L−<sup>1</sup> reducing sugars were released when 0.5 (2.80 FPU), 1 (5.60 FPU), 2 (11.2 FPU), and 4 mL (22.4 FPU) of Cellulase 17600 were used. These results suggest that an increasing of enzyme concentration in the reaction not necessarily imply higher hydrolysis of the neem seed. Therefore, it is advisable to use the lowest concentration in which the same results were obtained for each enzyme preparation. Crystalzyme Cran and Crystalzyme 100XL showed less hydrolysis of the neem seed after 18 h. Crystalzyme Cran released 1.0022 (± 0.0576) g L−<sup>1</sup> with the highest concentration of enzyme used (4 mL, 9.52 FPU); however, these results were not statistically different from those found when using 1 (2.38 FPU) and 2 mL (4.76 FPU), since the equilibrium concentration of reducing sugars was 0.9323 (± 0.0786) and 0.9859 (± 0.0341), respectively. On the other hand, the samples hydrolyzed with Crystalzyme 100XL had a maximum reducing sugar release when 4 mL of enzyme (21.2 FPU) was used.

In order to determine the optimum enzyme concentration to neem seed hydrolysis, equilibrium concentration and release rate of the reducing sugars were evaluated. The analysis of the results showed that reducing sugar release rate was not statistically different when 1, 2 or 4 mL for both Crystalzyme PML-MX and Cellulase 17600 L were used. When Crystalzyme Cran was evaluated, the higher rates of release of reducing sugars were obtained with 2 and 4 mL. In contrast, the highest reduced sugar release rate for Crystalzyme 100XL was presented when 1 mL (5.30 FPU) of this enzyme was added. Therefore, based on the analysis of results, it was considered that the units of activity necessary to hydrolyze the neem seed are 6.96, 5.60, 4.76, and 5.3 FPU mL−<sup>1</sup> for Crystalzyme PML-MX, Cellulase 17600 L, Crystalzyme Cran, and Crystalzyme 100XL, respectively.

After optimum conditions of pH, temperature and enzyme concentration were determined, and the kinetics of azadirachtin released was carried out but using fresh seed instead of dehydrated seed to minimize the risk of losses due to the dehydration process.

**Figure 2.** Optimum temperature of food-grade enzyme preparations: (A) Crystalzyme PML-MX, (B) Cellulase 17600,

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(C) Crystalzyme Cran, and (D) Crystalzyme 100XL.

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extract. A second reaction was carried out adjusting each hydrolysis to the optimum pH of each enzyme, and the effect of the temperature in the range of 25–70°C was evaluated (**Figure 2**).

Cellulase 17600L, Crystalzyme Cran, and Crystalzyme 100XL shown higher activity at 50°C, while to Crystalzyme PML-MX was at 45°C. These conditions are within the limit considered by [34] to maintain the neem extracts without the loss of azadirachtin. However, it is necessary to evaluate these conditions on the release of azadirachtin, since it is unknown whether the highest hydrolysis of the neem seed ensures maximum concentration of azadirachtin.

Results of the enzyme concentration effect over hydrolysis of neem seeds could be observed in **Figure 3**. Reducing sugar release was used as indirect measure of neem seed hydrolysis.

Reactions catalyzed by Crystalzyme PML-MX and Cellulase 17600 L presented the highest hydrolysis of the neem seed, probably due to the presence of cellulases in its composition. A

sugars were released when 0.5 (3.48 FPU), 1 (6.96 FPU), 2 (13.92 FPU), and 4 mL (27.84 FPU) of Crystalzyme PML-MX were used. On another hand, 1.8034 (± 0.0387), 2.0809 (± 0.0023), 1.9921

FPU), 2 (11.2 FPU), and 4 mL (22.4 FPU) of Cellulase 17600 were used. These results suggest that an increasing of enzyme concentration in the reaction not necessarily imply higher hydrolysis of the neem seed. Therefore, it is advisable to use the lowest concentration in which the same results were obtained for each enzyme preparation. Crystalzyme Cran and Crystalzyme 100XL showed less hydrolysis of the neem seed after 18 h. Crystalzyme Cran released 1.0022

results were not statistically different from those found when using 1 (2.38 FPU) and 2 mL (4.76 FPU), since the equilibrium concentration of reducing sugars was 0.9323 (± 0.0786) and 0.9859 (± 0.0341), respectively. On the other hand, the samples hydrolyzed with Crystalzyme 100XL

In order to determine the optimum enzyme concentration to neem seed hydrolysis, equilibrium concentration and release rate of the reducing sugars were evaluated. The analysis of the results showed that reducing sugar release rate was not statistically different when 1, 2 or 4 mL for both Crystalzyme PML-MX and Cellulase 17600 L were used. When Crystalzyme Cran was evaluated, the higher rates of release of reducing sugars were obtained with 2 and 4 mL. In contrast, the highest reduced sugar release rate for Crystalzyme 100XL was presented when 1 mL (5.30 FPU) of this enzyme was added. Therefore, based on the analysis of results, it was considered that the units of activity necessary to hydrolyze the neem seed are

After optimum conditions of pH, temperature and enzyme concentration were determined, and the kinetics of azadirachtin released was carried out but using fresh seed instead of dehy-

drated seed to minimize the risk of losses due to the dehydration process.

had a maximum reducing sugar release when 4 mL of enzyme (21.2 FPU) was used.

with the highest concentration of enzyme used (4 mL, 9.52 FPU); however, these

reducing sugars were released when 0.5 (2.80 FPU), 1 (5.60

for Crystalzyme PML-MX, Cellulase 17600 L, Crystalzyme

reducing

total of 1.8072 (± 0.0021), 2.0635 (± 0.0689), 2.0493 (± 0.0521), and 2.0742 (± 0.0283) g L−<sup>1</sup>

**3.3. Enzyme concentration**

116 Soil Contamination and Alternatives for Sustainable Development

(± 0.0456), and 1.9383 (± 0.0781) g L−<sup>1</sup>

6.96, 5.60, 4.76, and 5.3 FPU mL−<sup>1</sup>

Cran, and Crystalzyme 100XL, respectively.

(± 0.0576) g L−<sup>1</sup>

**Figure 2.** Optimum temperature of food-grade enzyme preparations: (A) Crystalzyme PML-MX, (B) Cellulase 17600, (C) Crystalzyme Cran, and (D) Crystalzyme 100XL.

**3.4. Azadirachtin release kinetics**

enized and adjusted at 1:10 (w v−<sup>1</sup>

encourage the azadirachtin degradation.

and 2.35 g kg−<sup>1</sup>

Evaluation of the azadirachtin release kinetics was conducted with 100 g of neem seed homog-

Biopesticide of Neem Obtained by Enzyme-Assisted Extraction: An Alternative to Improve the Pest Control

conducted under optimal conditions of each enzyme preparation, and azadirachtin were quantified at 0, 2, 4, 6, 12, 18, and 24 h by the HPLC technique, and the results were presented on base of the quantity of neem seed (dry base) used for the neem seed extract elaboration (**Figure 4**).

enzyme preparations were used, respectively. Both of this enzyme preparation include cellulases and pectinases and were statistically different (p < 0.05) from the enzymatic preparations,

Azadirachtin concentrations found in this study were higher than those reported by other authors when conventional methods such as extrusion, extraction with solvents (hexane, methanol), and aqueous extraction which reported concentrations of 1080, 565, 400, 150 ppm, respectively [33] and were similar to the obtaining by cold methanol extrusion [32]. However, the concentration obtained is lower than those reported with nonconventional technologies such as extraction with pressurized solvents, which reported concentrations of up to 9510 ppm of azadirachtin [34].

One of the advantages of the use of organic solvents in the extraction process is to improve the solubility of nonpolar components which increase their extraction from vegetable matrices. However, in the present study, only phosphate buffer was used, which could explain the differences of azadirachtin extraction with the pressurized solvent method [34]. Also, in [43], it was reported that in the tropical regions, there are lower concentrations of azadirachtin after extraction processes due to high temperatures, moisture, and storage conditions that could

(2350 ppm) neem seed when Cellulase 17600 L and Crystalzyme PML-MX

The highest azadirachtin concentrations obtained in this study were 2.55 g kg−<sup>1</sup>

Crystalzyme Cran (pectinases) and Crystalzyme 100XL (pectinases and arabinases).

**Figure 4.** Azadirachtin release kinetics from neem seeds extracted with food-grade enzyme preparations.

) ratio with phosphate buffer. Enzymatic hydrolysis was

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(2550 ppm)

119

**Figure 3.** Optimum filter paper unit (FPU) of the food-grade enzyme preparations for hydrolysis of neem seeds. (A) Crystalzyme PML-MX, (B) Cellulase 17600, (C) Crystalzyme Cran, and (D) Crystalzyme 100XL.

### **3.4. Azadirachtin release kinetics**

Evaluation of the azadirachtin release kinetics was conducted with 100 g of neem seed homogenized and adjusted at 1:10 (w v−<sup>1</sup> ) ratio with phosphate buffer. Enzymatic hydrolysis was conducted under optimal conditions of each enzyme preparation, and azadirachtin were quantified at 0, 2, 4, 6, 12, 18, and 24 h by the HPLC technique, and the results were presented on base of the quantity of neem seed (dry base) used for the neem seed extract elaboration (**Figure 4**). The highest azadirachtin concentrations obtained in this study were 2.55 g kg−<sup>1</sup> (2550 ppm) and 2.35 g kg−<sup>1</sup> (2350 ppm) neem seed when Cellulase 17600 L and Crystalzyme PML-MX enzyme preparations were used, respectively. Both of this enzyme preparation include cellulases and pectinases and were statistically different (p < 0.05) from the enzymatic preparations, Crystalzyme Cran (pectinases) and Crystalzyme 100XL (pectinases and arabinases).

Azadirachtin concentrations found in this study were higher than those reported by other authors when conventional methods such as extrusion, extraction with solvents (hexane, methanol), and aqueous extraction which reported concentrations of 1080, 565, 400, 150 ppm, respectively [33] and were similar to the obtaining by cold methanol extrusion [32]. However, the concentration obtained is lower than those reported with nonconventional technologies such as extraction with pressurized solvents, which reported concentrations of up to 9510 ppm of azadirachtin [34].

One of the advantages of the use of organic solvents in the extraction process is to improve the solubility of nonpolar components which increase their extraction from vegetable matrices. However, in the present study, only phosphate buffer was used, which could explain the differences of azadirachtin extraction with the pressurized solvent method [34]. Also, in [43], it was reported that in the tropical regions, there are lower concentrations of azadirachtin after extraction processes due to high temperatures, moisture, and storage conditions that could encourage the azadirachtin degradation.

**Figure 4.** Azadirachtin release kinetics from neem seeds extracted with food-grade enzyme preparations.

**Figure 3.** Optimum filter paper unit (FPU) of the food-grade enzyme preparations for hydrolysis of neem seeds.

(A) Crystalzyme PML-MX, (B) Cellulase 17600, (C) Crystalzyme Cran, and (D) Crystalzyme 100XL.

118 Soil Contamination and Alternatives for Sustainable Development

Although the highest concentrations of azadirachtin were obtained with the enzymatic preparations, Cellulase 17600L and Crystalzyme PML-MX, also Crystalzyme Cran and Crystalzyme 100XL could be used to elaborate neem extracts because of their azadirachtin concentrations of 1540 and 1690 ppm, respectively, are similar to those contained in commercial acaricidal products elaborated based on neem, but with the use of solvents [32]. This result indicates an advantage and a possible solution to the indiscriminate use of synthetic pesticides and the organic solvents employed in several extractions of active biomolecules. On the other hand, after 18 h of enzymatic hydrolysis, there are no changes on azadirachtin release, which means it is time required to carry out the obtaining of neem seed extracts. In addition, the conditions identified in this study as necessary to neem seed hydrolysis are not extreme or aggressive and could be easily scalable.

**Conflict of interest**

to declare.

**Author details**

Argel Flores Primo<sup>1</sup>

María D. Marriezcurrena2

Sóstenes R. Rodríguez1

State of Mexico, Mexico

10.1007/978-3-319-27455-3

DOI: 10.1016/S0013-9351(03)00112-9

10751132\_14

**References**

\*, Violeta T. Pardío1

\*Address all correspondence to: mopri02@yahoo.com.mx

, Arfaxad Aguilar1

All authors who participate in the elaboration of this manuscript have no conflict of interest

Biopesticide of Neem Obtained by Enzyme-Assisted Extraction: An Alternative to Improve the Pest Control

, Karla M. López<sup>1</sup>

1 Faculty of Veterinary Medicine and Zootechnics, University of Veracruz, Veracruz, Mexico

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2 Faculty of Agricultural Science, Autonomous University of the State of Mexico,

, Elissa Chávez1

, Dora L. Pinzón2

and

,

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