**2. Assisted enzymatic extraction of neem**

The study was divided into two stages: (1) determination of optimum activity conditions of four enzyme preparations and (2) evaluation of the azadirachtin release kinetics under optimal conditions of enzyme preparation activities. The enzyme preparations used in this study were Crystalzyme PML-MX, Cellulase 17600, Crystalzyme Cran, and Crystalzyme 100XL, and their optimum pH and temperature conditions were identified by using dehydrated and pulverized neem seed. Then, four volumes (1, 2, 3, and 4 mL) of each enzyme were tested to determine the optimum enzyme concentration to neem seed hydrolysis.

Evaluations of azadirachtin release kinetics were conducted at optimum pH and temperature for each enzyme preparation, and the maximum time required for the azadirachtin extraction was determined.

#### **2.1. Material**

Neem seeds were provided by a local producer from Jamapa, Veracruz during June, 2017. One kilogram of 110–120 day old neem seeds (green-yellow coloration) was collected. Harvested seeds were washed and cut in half to extract the cotyledon extraction, which was stored by in freezing at −20°C for 24 h before using.

#### **2.2. Food-grade enzyme preparation cellulolytic activity**

Each preparation enzymatic activity was measured by filter paper test [39], where the activity was defined as filter paper units per enzyme milliliter (FPU. mL−<sup>1</sup> ). After FPU. mL−<sup>1</sup> determination, the effect of the enzyme concentration on the hydrolysis of the neem seed cellulose structures and the azadirachtin release was evaluated. Neem seed hydrolysis was indirectly determined by the quantification of reducing sugar released during the enzymatic reaction. Total protein was estimated with [40] bovine serum albumin (BSA) as standard, and enzymatic activity (EA) for each enzymatic preparation was defined as changes in absorbance in 0.001 of reducing sugars mg protein−<sup>1</sup> min−<sup>1</sup> .

#### **2.3. Optimum conditions of pH, temperature, and enzyme concentration**

The optimal conditions of pH and temperature were established based on the enzymatic hydrolysis of the neem seed and the release of azadirachtin. To determine the optimum pH of the enzymatic preparations, 1 g of the dehydrated seed was homogenized with 19 mL of phosphate buffer (3.0, 3.5, 4.0, 4.5, 5.0, and 5.5) and was conditioned at 50°C for 5 min and after 1 mL of the corresponding enzyme preparation was added. The enzymatic reaction was carried out for 2 h and was stopped by immersion in ice water. Subsequently, 1 mL of the sample was taken for reducing sugar determination and 1 mL for azadirachtin quantification by HPLC [41]. Once the optimum pH of each enzyme preparation was identified, the effect of the temperature at 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70°C was evaluated. Additionally, one extract without enzyme added was prepared as control, and the concentrations of reducing sugars released were subtracted from the values obtained in each of the enzymatic treatments. Furthermore, enzyme:neem seed (dry base) ratio was evaluated under optimal conditions of pH and temperature, and for this, 2 g of neem seeds (wet base) were used and 0.5, 1.0, 2.0, and 4 mL of each of the enzyme preparations were tested.

#### **2.4. Kinetics of azadirachtin release**

of azadirachtin from neem depends on several factors as age of the tree, region of its production, stage of fruit development, availability of the internal portion of the seed, storage conditions of the seed, methods and solvents used for its extraction, and the particle size [32–35]. However, one aspect that has not been taken into account is the physical barrier exerted by the cell wall of the seed that directly affects the availability and extraction of azadirachtin and other acaricidal compounds. This problem has been overcome with great success in the plant extract industry through the application of cellulases or preparations with multiple enzy-

The assisted extraction by cellulolytic enzymes has proven to be a viable and feasible tool to obtain bioactive metabolites from plants, due to its effect on lignocellulosic structures of its cell wall, which increase the yield of oils, pigments, flavorings, and aromas extracted in comparison with traditional extraction methods [38]. Due to the advantages of the use of cellulolytic enzymes for the production of bioactive metabolites from plants and the inherent need to develop sustainable and environmentally friendly alternatives that avoid the resistance phenomenon, the present study had the purpose of evaluating the use of food-grade enzyme preparation on the hydrolysis of the neem seed to obtain extracts with higher concentrations

The study was divided into two stages: (1) determination of optimum activity conditions of four enzyme preparations and (2) evaluation of the azadirachtin release kinetics under optimal conditions of enzyme preparation activities. The enzyme preparations used in this study were Crystalzyme PML-MX, Cellulase 17600, Crystalzyme Cran, and Crystalzyme 100XL, and their optimum pH and temperature conditions were identified by using dehydrated and pulverized neem seed. Then, four volumes (1, 2, 3, and 4 mL) of each enzyme were tested to

Evaluations of azadirachtin release kinetics were conducted at optimum pH and temperature for each enzyme preparation, and the maximum time required for the azadirachtin extraction

Neem seeds were provided by a local producer from Jamapa, Veracruz during June, 2017. One kilogram of 110–120 day old neem seeds (green-yellow coloration) was collected. Harvested seeds were washed and cut in half to extract the cotyledon extraction, which was stored by in

Each preparation enzymatic activity was measured by filter paper test [39], where the

determination, the effect of the enzyme concentration on the hydrolysis of the neem seed

mL−<sup>1</sup>

). After FPU.

mL−<sup>1</sup>

matic activities (cellulases, hemicellulases, and pectinases) [34, 36, 37].

determine the optimum enzyme concentration to neem seed hydrolysis.

of azadirachtin, but without the use of solvents.

112 Soil Contamination and Alternatives for Sustainable Development

**2. Assisted enzymatic extraction of neem**

was determined.

freezing at −20°C for 24 h before using.

**2.2. Food-grade enzyme preparation cellulolytic activity**

activity was defined as filter paper units per enzyme milliliter (FPU.

**2.1. Material**

To determine kinetics of azadirachtin release, 100 g of neem seed (wet base) was homogenized with phosphate buffer at the optimum pH previously identify (1:10, w v−<sup>1</sup> ) ratio for 3 min using an Ultra Turray homogenizer, T-25 basic (IKA®, Wilmington, NC). Five extracts were elaborated, one for each enzyme preparation and one without enzyme (control), and were incubated at optimum temperatures for each one. An aliquot was taken at 0, 2, 4, 6, 12, 18, and 24 h for the determination of azadirachtin. All samples were analyzed by triplicate.

#### **2.5. Determination of azadirachtin**

Azadirachtin quantification was carried out by the HPLC technique proposed by [41], using a binary HPLC system (Waters 1525) and a photodiode detector (Waters 2996). The analytical separation was carried out with a Nova-Pak C18 column of 4 μm (3.9 × 150 mm) SUM (Waters® Milford, MA). Neem samples were centrifuged and diluted with acetonitrile (1:1, v v−<sup>1</sup> ) before analyzing by HPLC. The samples were filtered through acrodiscs (Millipore) of 0.22 μm, and 20 μl was injected into the column. The flow rate was set at 1 mL min−<sup>1</sup> , the mobile phase was acetonitrile: water (40:60, v v−<sup>1</sup> ), and azadirachtin was read at 217 nm in a retention time of 3.1 min.

#### **2.6. Statistical analysis**

The optimal conditions of pH, temperature, and the enzyme:substrate ratio were analyzed by means of analysis of variance (ANOVA) at a level of significance of p < 0.05, and Tukey's tests 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 firstorder empirical equation Yi = Ye (1−e−kt), in which the equilibrium concentrations (Ye ) and its release rate constant (*k*) were determined, and the results were analyzed by ANOVA (p < 0.05) to determine differences between treatments.
