Optimization of hydrolysis conditions for release of biopesticides from glucosinolates in Brassica juncea and Sinapis alba seed meal extracts

Industrial Crops and Products 97 (2017) 354–359

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Optimization of hydrolysis conditions for release of biopesticides from glucosinolates in Brassica juncea and Sinapis alba seed meal extracts Inna E. Popova ∗ , Jeremiah S. Dubie, Matthew J. Morra Department of Plant, Soil, and Entomological Sciences, University of Idaho, 875 Perimeter Drive, MS 2339, Moscow, ID 83844-2339, USA

a r t i c l e

i n f o

Article history: Received 28 September 2016 Received in revised form 6 December 2016 Accepted 22 December 2016 Keywords: Biopesticide Glucosinolates Mustard Myrosinase Oilseed

a b s t r a c t Development of alternative, economically sound methods of pest control is a priority in modern agriculture due to the rise in pest resistance and increased demand for organic crops. Although Brassicaceae seed meals have demonstrated promise as natural pesticides, use is limited by the bulky nature of the material and inconsistent efficacy. Here is presented an optimization of hydrolysis conditions for production of pesticidal compounds from extracts of yellow (Sinapis alba L.) and Oriental (Brassica juncea L.) mustards. Upon hydrolysis by the endogenous enzyme myrosinase, glucosinolates sinigrin and sinalbin naturally present in mustard produce biologically active 2-propenyl isothiocyanate and SCN− , respectively. Hydrolysis solution composition, time, and amount of mustard seed meal as a source of myrosinase were optimized to result in maximum production of 2-propenyl isothiocyanate from B. juncea and SCN− from S. alba extracts. Using optimized conditions, the release of active pesticidal compounds from the extract can be achieved within 48 h. Formulated extracts can be prepared in such a manner that they will be potentially suitable for organic certification. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The development of alternative, economically sound methods of pest control is a priority in modern agriculture due to a rise in pest resistance and increased demand for organic crops (Shaner, 2014). It is estimated that more than 471 weed species demonstrate resistance to commonly used herbicides such as those in the imidazolinone, sulfonylurea, and triazolopyrimidine sulfonanilide families. In addition, more than 500 species of insects are documented to have resistance to at least one pesticide (Ferguson et al., 2001; Heap, 2014; Hoy, 1998). Meals of yellow (Sinapis alba L.) and Oriental (Brassica juncea L.) mustards are potential alternatives to synthetic pesticides for controlling weeds, nematodes, insects, and fungi in field and greenhouse settings (Avato et al., 2013; Boydston et al., 2011; Larkin and Griffin, 2007; Yu and Morishita, 2014) as a consequence of contained glucosinolate substrates that are enzymatically hydrolyzed by myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147) to produce a variety of biologically active products (Clarke, 2010). Mustard seed can be cold pressed to produce oil for biodiesel (Peterson et al., 2006) and high protein seed meals that retain intact glucosinolates and myrosinase.

∗ Corresponding author. E-mail address: [email protected] (I.E. Popova). http://dx.doi.org/10.1016/j.indcrop.2016.12.041 0926-6690/© 2016 Elsevier B.V. All rights reserved.

There are challenges associated with the use of mustard seed meals as pesticides including batch-to-batch variability resulting from unpredictability in mustard growing, processing, and storage conditions. Other challenges are associated with the cost and logistics of transportation, storage, and application of mustard meal, as typical field application rates for mustard meal are relatively high (3.2–14.5 g m−2 ) (Boydston et al., 2011). Introduction of seed meal at these rates results in organic carbon (80% organic carbon by weight) and nitrogen loads (approximately 5% by weight) to the field that may create adverse or unexpected effects such as the growth of undesirable organisms (Rice et al., 2007). To overcome these challenges, glucosinolate-containing extracts from mustard seed meal can be used as a source of biopesticidal hydrolysis products. Glucosinolates constituting up to 10% of total mustard meal weight can be extracted with aqueous methanol (Cools and Terry, 2012). Glucosinolates themselves are biologically inactive and can be preserved in extracts for prolonged amounts of time (unpublished data). In the presence of water, myrosinase catalyzes glucosinolate hydrolysis and the release of biologically active compounds. The major glucosinolate in S. alba, sinalbin, is hydrolyzed to an unstable isothiocyanate that non-enzymatically produces SCN− , a phytotoxic compound (Fig. 1) (Borek and Morra, 2005). Sinigrin, a major glucosinolate in B. juncea, is hydrolyzed to produce volatile and bioactive 2-propenyl isothiocyanate (Dai and Lim, 2014).

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355

HO OH HO

OH OH OH

N HO

S

Myrosinase H2O

HO

HO

OH

+ SO42-+ SCN-+ 2 H3O+

+

O

OH HO

O

OH

O S

O

O

Sinalbin

CH2

OH

OH OH

HO

OH N HO

S

Myrosinase H2O

O

OH

HO

+

+ SO42-+ H3O+

OH HO

N

O

C

O

S

S O

CH2

O

Sinigrin Fig. 1. Enzymatic hydrolysis products of two major mustard glucosinolates, sinalbin and sinigrin.

Hydrolysis of mustard extracts can be performed prior to field application. For example, extracts from yellow mustard containing sinalbin can be hydrolyzed to yield a stable SCN− solution. This solution can be applied through existing sprinkler or sprayer systems for control of weeds. Alternatively, hydrolysis of mustard extracts can be performed on-site at the point of pest control when the extract is applied to the field. This approach is particularly useful for controlling pests such as nematodes with 2-propenyl isothiocyanate, a volatile hydrolysis product of sinigrin present in Oriental mustard (Larkin and Griffin, 2007). As a result, the use of extracts instead of mustard meals would reduce transportation costs, make application compatible with spray systems, and allow more precise control of active ingredient concentration in the final product. Although seed meal extracts have advantages, hydrolysis of the contained glucosinolates requires formulation of the extract with adequate enzyme and buffering of pH within a range required for enzymatic activity. The objective was to optimize hydrolysis conditions for sinigrin and sinalbin in meal extracts. Hydrolysis solution composition, time, and amount of mustard seed meal used as a source of myrosinase were optimized to result in maximum production of 2-propenyl isothiocyanate and SCN− from B. juncea and S. alba seed meal extracts, respectively. 2. Materials and methods 2.1. Materials Mustard seeds and meals of S. alba (IdaGold) and B. juncea (Pacific Gold and Kodiak) were obtained locally (Latah County, ID, USA). Oil contents of seeds and meals were analyzed gravimetrically after extraction with hexane. A sinigrin (2-propenyl glucosinolate) standard and 2-propenyl isothiocyanate were purchased from Sigma-Aldrich (St. Louis, MO, USA). A sinalbin standard was isolated from S. alba. Acetonitrile, water, methanol, and other solvents were of HPLC or LC/MS grade. Solvents and all other chemicals (at least of analytical grade) were purchased from Sigma–Aldrich or Thermo Fisher (Pittsburgh, PA, USA).

2.2. Mustard meal extract preparation Mustard meal with intact glucosinolates and myrosinase was obtained from a cold press facility (University of Idaho, Moscow, ID) as previously documented (Brown et al., 1991). For myrosinase studies, Kodiak seed was ground in a coffee grinder with minimal heat generation to avoid myrosinase denaturation. Mustard meals were homogenized and passed through a 2-mm sieve. Mustard meal was extracted with 73% (v/v) methanol at a 1:20 v/v ratio using an end-to-end shaker at room temperature for 2 h. Seed debris was separated by filtering, and filtrates were concentrated by rotary evaporation to remove most of the solvent. Concentrated extract was then freeze-dried to obtain a free flowing powder with a particle size of <45 ␮m. The concentration of sinalbin in the S. alba meal extract was 777 ␮mol g−1 and the concentration of sinigrin in the B. juncea meal extract was 555 ␮mol g−1 . 2.3. Hydrolysis of glucosinolates in mustard seed meal extracts Hydrolysis of glucosinolates in mustard meal extracts was performed by adding mustard meal of the same species to the extracted powder and statically incubating the aqueous slurry in closed glass vials at room temperature. Hydrolysis optimization was performed using 0.1 g of mustard meal with 0.05–0.3 g of mustard meal extract in 2.5 mL of aqueous solution. Hydrolysis media were modified with buffers (different pHs and concentrations) and ascorbate. Time of hydrolysis was optimized from 30 min to 48 h, and hydrolysis efficiency was calculated as the percentage of intact glucosinolate dissipation during 24 h. All reported glucosinolate concentrations and dissipation percentages include total glucosinolate in both the extract and seed meal. 2.4. Derivatization of 2-propenyl isothiocyanate An aliquot (10–100 ␮L) of the hydrolysis mixture was obtained through a septum of the capped glass reaction vial and diluted with methanol to 5 mL. Diluted solution (860 ␮L) was added to

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a 2-mL autosampler vial containing 860 ␮L of 100 mM potassium phosphate at pH 8.5. An additional 280 ␮L of 35 mM 1,2-benzenedithiol/1% mercaptoethanol in methanol was added, and the vial was capped and incubated for 1 h at 65 ◦ C. After incubation, the mixture was vortexed, centrifuged at 800 g, and analyzed by HPLC/UV. Validation of analyte recovery was confirmed using aqueous samples spiked with 2-propenyl isothiocyanate. 2.5. HPLC/UV analysis of derivitized 2-propenyl isothiocyanate Analysis of derivitized 2-propenyl isothiocyanate was performed using an Agilent 1200 Series HPLC system with a diode array detection (DAD) system on an Agilent XDB C18 (1.8 ␮m, 4.6 × 50 mm) column (Agilent, Santa Clara, CA, USA). The column was thermostated at 30 ◦ C. Isocratic elution was used with 90% acetonitrile in water and the flow rate was 0.6 mL min−1 . Spectra were recorded from 190 to 400 nm in 2-nm steps. The injection volume was 5 ␮L and the runtime was 5 min with derivitized 2propenyl isothiocyanate elution at 1.4 min. Derivitized 2-propenyl isothiocyanate was quantified at an extracted wavelength channel of 350–360 nm using an external calibration curve. 2.6. Ion chromatographic analysis Sinigrin, sinalbin, sulfate, and SCN− in the extracts were quantified by ion chromatography (IC) using a Dionex Ion Analyzer equipped with a GP40 gradient pump, ED40 electrochemical detector, and an AS40 autosampler as described previously (Popova and Morra, 2014). Briefly, a Dionex 4 × 210 mm Ion-Pac AS16 anion exchange column was used for separation. Sodium hydroxide (100 mM) was used as the mobile phase at a flow rate of 0.9 mL min−1 . The detector stabilizer temperature was set at 30 ◦ C with temperature compensation of 1.7% per ◦ C. Anion suppressor current was set to 300 mA. The injection volume was 20 ␮L. 2.7. Data analysis Experiments were performed in duplicate or triplicate and are presented as means ± one standard deviation. Significant differences among analyte concentrations detected by different methods of analysis were determined using one-way analysis of variance (ANOVA) with a p < 0.05 level of significance. All analyses were performed using JMP software (version 10, SAS Institute Inc., Cary, NC, USA). 3. Results and Discussion 3.1. Mustard seed meal as a source of myrosinase During extraction with 73% aqueous methanol endogenous myrosinase is deactivated and glucosinolates are extracted intact and unhydrolyzed. As a consequence, a source of active myrosinase must be reintroduced to the extract to facilitate glucosinolate hydrolysis. A variety of mustard seeds and seed meals were therefore evaluated as to their myrosinase activities in order to assure adequate glucosinolate hydrolysis efficiency. Multiple means are available to monitor reaction progress including sulfate production, sinigrin disappearance, and the drop in pH that occurs as a result of the hydrolytic reaction shown in Fig. 1. A typical experiment in which the myrosinase source (0.1 g crushed whole seed or partially defatted seed meal) was combined with B.juncea extract (0.3 g) showed that after 3 d sinigrin hydrolysis efficiency varied from 33 to 98% depending on whether seed or meal was used, the length of time seed and meal were stored, and the meal defatting method (Fig. 2).

Fig. 2. Relative difference in myrosinase activity in B. juncea seed and seed meals (Kodiak (K) or Pacific Gold (PG)) as determined by quantifying one of the reaction products sulfate and the remaining sinigrin substrate. Reaction pH was determined after incubating 0.3 g extract with 0.1 g of seed or seed meal for 3 d.

While myrosinase is relatively stable in intact seeds, activity may decline up to 99.5% when seeds are processed (Bell and Keith, 1991). The decline in activity can be attributed to thermal deactivation by localized heat generated during seed pressing since even a rise of temperature to 60 ◦ C for less than 10 min can lead to inactivation of 20% of the available myrosinase (Veto-Kiszter et al., 2009). Care was taken in seed pressing and grinding of samples used here to avoid thermal deactivation of myrosinase. The time period that meal is stored also affects myrosinase activity (Fig. 2). In these experiments, hydrolysis efficiency of B. juncea seed meal myrosinase was reduced from 91% to 52% with 2 years of storage. Defatted meal stored for 2 years retained only 33% of its hydrolysis efficiency. It is of note that a three-fold decline in myrosinase activity did not reduce hydrolysis rates of glucosinolates present at endogenous seed meal concentrations (data not show), however preliminary investigations demonstrated that myrosinase activity was limiting when combined with extracts in which glucosinolate concentrations were 4 to 5 times higher than those in mustard seed meal. Thus, mustard meal intended for use as the enzyme source for hydrolysis of glucosinolates in extracts should be evaluated to ensure that sufficient myrosinase activity is present to release hydrolysis products within the required time frame. For optimization experiments described below, seed meals with near maximal hydrolysis efficiencies as compared with efficiencies measured in seed stored for short time periods were used (Fig. 2). Although a high concentration of myrosinase is desired for rapid hydrolysis, the amount of seed meal added as the enzyme source has other consequences. One significant issue is formation of a thick paste when the amount of meal used for hydrolysis exceeds 20% of the weight of water used as a solvent. All the added water is essentially incorporated into the mucilaginous structure of the seed meal, and no aqueous phase can be separated and recovered as indicated by the low recovery of SCN− for the 5:1 solvent to meal + extract ratio (Fig. 3). Mustard meal has a relatively large mucilage content (up to 5% by weight of seed), which is a mixture of a pectic material consisting primarily of galacturonic acid, galactose, rhamnose, and a 1,4-linked ␤-d-glucan (Balke and Diosady, 2000; Cui et al., 1993). In aqueous media, mucilage expands when hydrated to form hydrocolloids (Gerhards and Walker, 1997). As a consequence of elevated mucilage content, volume of the recoverable solvent is reduced and viscosity of the solution is increased (Cui et al., 1993). Extraction efficiencies were directly proportional to the solvent to seed meal

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357

Glucosinolate hydrolyzed (%)

Sinigrin

Sinalbin

100 90 80 70 60 50 40 no buffer

Fig. 3. Optimization of solvent (H2 O) to extract + meal ratio (w:w) required for hydrolysis of sinalbin in S. alba extracts as determined by incubating 0.3 g extract with 0.2 g seed meal and quantifying SCN− production after 24 h (n = 2).

75

4.0

45

3.0

30

2.0

15

1.0 0.0

0

18

31

45

72

85

Unidentified product Sinigrin 2-Propenyl isothiocyanate

50

7.5 6.5 5.5

40

4.5

30

3.5

pH

Reactant or product (mM)

Initial sinalbin (mM)

60

2.5

20

1.5 10

0.5

0

-0.5 5

12

19

6.6

6.9

7.2

7.5

pH Fig. 5. Hydrolysis of S. alba and B. juncea mustard extracts (0.15 g) in the presence of mustard meal (0.1 g) in 2.5 mL of 200 mM phosphate buffer (n = 3).

5.0

60

5

6.3

6.0

Unidentified product Sinalbin SCN-

pH

Reactant or product (mM)

90

6.0

26

40

47

Initial sinigrin (mM) Fig. 4. Hydrolysis of S. alba and B. juncea mustard extracts (0.05-0.3 g) incubated for 24 h in the presence of mustard meal (0.1 g) and 2.5 mL of water. The line represents pH of the reaction solutions after 24 h of hydrolysis (n = 2).

ratios from 10:1 to 50:1, reaching 130 ␮mol (Fig. 2). Approximately 120 ␮mol of SCN− was recovered with a 25:1 ratio, producing an extract with approximately twice the concentration of SCN− as compared with a 50:1 ratio. Lower solvent to seed meal ratios can be used to produce extracts with higher SCN− concentrations, but the decrease in extraction efficiency was deemed unacceptable. A 25:1 solvent to seed meal ratio was chosen for the following experiments to optimize extraction efficiency and minimize solvent use. 3.2. Optimization of hydrolysis pH and buffering Increasing the amount of mustard extract relative to the meal resulted in incomplete hydrolysis (Fig. 4). Residual sinalbin was

observed when the initial sinalbin concentration reached 45 mM and residual sinigrin at only 19 mM, most likely indicating the higher myrosinase concentration reported to occur in S. alba seed as compared with B. juncea seed (Kumar et al., 2011). A myrosinase limitation was also indicated by the fact that despite an increase of sinalbin extract added to the reaction mixture, the maximum concentration of SCN− produced peaked at 24 mM, a concentration about five times higher than could be produced from the original mustard meal. Similarly, the maximum concentration of 2-propenyl isothiocyanate produced was 14 mM even when up to 47 mM of sinigrin was added to the meal in the form of a mustard extract (Fig. 4). Hydrolysis of glucosinolates in mustard extracts is limited by the production of hydronium ion (Fig. 1) as reflected in the drop in pH that occurs with glucosinolate hydrolysis (Fig. 4). Sinigrin is enzymatically hydrolyzed to equimolar amounts of 2-propenyl isothiocyanate, sulfate, glucose, and hydronium ion. Sinalbin is enzymatically hydrolyzed to equimolar amounts of 4hydroxybenzyl alcohol, SCN− , sulfate, glucose, and two moles of hydronium ion. The release of hydronium ion shifts the pH to a more acidic region. Upon hydrolysis of endogenous glucosinolates, pH typically decreases by one unit from 5.8 to 4.6, at which point myrosinase activity is still adequate. Myrosinase has maximal activity at pHs of 5-7, while its activity is almost negligible at low pH (Iori et al., 1996). However, when mustard extracts are added to meal, more than a three-fold increase in the glucosinolate concentration results in a greater and more rapid pH drop. Hydrolysis of sinalbin at concentrations four times higher than endogenous concentrations resulted in a pH of 2.5 in 24 h and consequently, myrosinase inhibition (Fig. 4). A drop in pH in sinigrin hydrolysis mixtures to 4.6 in 24 h was sufficient to eliminate detectable activity because of the lower myrosinase concentration in B. juncea seed meal as compared with S. alba seed meal (Kumar et al., 2011). Lower pHs also favor nitrile production (Bellostas et al., 2008; Borek et al., 1994), possibly accounting for the unidentified product (Fig. 4). This excess of glucosinolate relative to the meal thus leads to a change in pH that exceeds buffering capacity of that meal. To prevent inhibition of myrosinase by increased acidity, a series of phosphate buffers in the pH range from 6.0 to 7.5 was used instead of water for glucosinolate hydrolysis (Fig. 5). When 200 mM phosphate buffer was used, more than 90% of the sinigrin and sinalbin were hydrolyzed in the pH range from 6.3 to 7.2, while some of the glucosinolate was still unhydrolyzed when pH was increased to 7.5. The minimum concentration of phosphate buffer required for maintaining pH as expressed on a molar basis was 1.5–2 times

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120

SCN¯ (%)

100 80 60 40

Water 150 mM Bicarbonate 150 mM Phosphate buffer (pH 6.5)

20 0 0

6

12

18

24

30

36

Time (h) Fig. 6. Effect of ascorbate addition on the production of 2-propenyl isothiocyanate and SCN− from B. juncea and S. alba mustard powder. Mass balance closure represents the percent of glucosinolate converted to biologically active 2-propenyl isothiocyanate and SCN− on a molar basis (n = 3).

9 8 7

sinalbin concentration and equal to sinigrin concentration, thus supporting reaction stoichiometry (Fig. 1).

Although sinigrin and sinalbin were completely hydrolyzed in buffered media with mustard meal as the myrosinase source (Fig. 5), only 87-92% of expected biologically active hydrolysis products were produced (Fig. 6). To achieve quantitative conversion of intact glucosinolates to their biologically active products, hydrolysis media composition was further optimized. While the major products of sinalbin and sinigrin hydrolysis are SCN− and 2-propenyl isothiocyanate, respectively, other products may be formed as well. For example, in the presence of Fe2+ under acidic conditions, nitriles and thioamides can form (Bellostas et al., 2008). To increase the desirable product yield, ascorbic acid was added to the reaction mixture. The concentration of ascorbic acid added to solution was tested between 0.1 and 100 mM (Fig. 6). Ascorbic acid acts as a co-factor for myrosinase, enhancing the production of SCN− and 2-propenyl isothiocyanate (Burmeister et al., 2000; Nagashima and Uchiyama, 1959). For example, it was demonstrated previously that addition of ascorbic acid increased 2-propenyl isothiocyanate production from mustard meal by 18% (Sharma et al., 2012). While ascorbic acid is naturally present in mustard meal at concentrations as high as 80 ␮g g−1 , this concentration may not be sufficient for hydrolysis of mustard extracts (Shikita et al., 1999). Indeed, when 1–25 mM of ascorbic acid was added to the hydrolysis solution, 98% of sinalbin was converted to SCN− , and 99% of sinigrin was converted to 2-propenyl isothiocyanate (Fig. 6). 3.4. Optimization of hydrolysis time The final step in optimization of mustard meal extract hydrolysis was optimization of hydrolysis time. In natural systems, the glucosinolate-myrosinase system is part of a plant defense mechanism (Hopkins et al., 2009). When mustard plant tissue is damaged in the presence of water, hydrolysis reactions are initiated and hydrolysis products are immediately released. When mustard extract is hydrolyzed under static conditions, hydrolysis is limited by the diffusion of substrate from extract to the enzyme in mustard meal and diffusion of products from the enzyme active site to the bulk solution. Thus, additional time is required to allow for complete hydrolysis of glucosinolates due to the significantly higher concentrations of glucosinolates and a larger volume of solvent.

pH

3.3. Optimization of hydrolysis media composition

6 5 4 3

Water 150 mM Bicarbonate 150 mM Phosphate buffer (pH 6.5)

2 1 0 0

6

12

18

24

30

36

Time (h) Fig. 7. Hydrolysis of S. alba mustard extracts (0.15 g) in the presence of mustard meal (0.1 g) in 2.5 mL of 150 mM phosphate buffer or 150 mM potassium bicarbonate (n = 3).

Under static conditions with phosphate buffer at a final concentration of 150 mM, complete hydrolysis of sinalbin was observed in 24 h (Fig. 7). Based on the hydrolysis studies presented in the manuscript, it is expected that both mustard extracts would exhibit similar behavior. Stability and the lack of SCN− volatility allowed us to monitor pH, whereas accomplishing these same studies with volatile and potentially labile 2-propenyl isothiocyanate would have led to inaccurate results. While phosphate buffers are efficient buffers, their use may be undesirable for some applications or the cost prohibitive. To address such concerns, carbonate and bicarbonate were considered as alternative buffering agents. It was demonstrated that at the same concentration (1.5–2 times the expected concentration of glucosinolates in the extracts) both carbonate and bicarbonate were equally efficient in maintaining hydrolysis mixture pH at 6.5, providing complete hydrolysis of sinalbin and sinigrin. One of the advantages of using carbonate buffer for pH adjustment is that carbonate can be obtained from natural sources, thus making the final product potentially eligible for organic certification. Phosphate buffer allows for faster hydrolysis, and sinalbin and sinigrin can be hydrolyzed under static conditions in just 12 h because the initial pH of 6.5 coincides with the optimum pH for myrosinase (Fig. 7). When potassium bicarbonate was used for maintaining pH, the initial pH of 9.5 decreased to 6.5 with hydrolysis as hydronium ions were released from glucosinolates. Since myrosinase activity at pH 9.5 is lower than that at pH 6.5, initial hydrolysis reaction rates were slower as compared with the phosphate-buffered systems. When no buffering agent was used,

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