117 6 2 4–6 The 13,14 6 In India While the 13,14 12 . 6 - mono-, myo ), through the release 6 -inositol with less myo is a negatively charged compound 6 are negligible in households that regularly 6 . 1 − inositol hexaphosphate (InsP mol g 𝛍 myo- - 0.05) after 24 h of activation, but only represents a 4.75% degradation into lower Correspondence to: AE Mitchell, Department ofUniversity Food of Science California, Davis, and CA Technology, 95616, USA. E-mail: [email protected] Department of Food Science andCA, USA Technology, University of California, Davis, D 6 One of the most commonly used methods used to remove phy- < ∗ tate from cereals, grains, and legumes is to soak them in water. Soaked products may be differentiated basedperature on and the pH tem- of the soak water,The and the most length of common soaking time. forms of soaked include sprouted, becomes significant for individuals that rely on -based diets. Soaking is an ancient practiceenhance in the many cultures nutritional intended value to and/or flavor of food. consume meat, reduced due to InsP bioavailability by forming insolublewith salts positively and charged complexes minerals infound pH in environments food commonly and the human digestive system (pH 4–8). adverse effects of InsP and Pakistan, raw almonds arepart soaked of overnight a and meal served to as promote cognitive a development in children. majority of these complexes remain insoluble duringthe digestion, human as stomach and smallphytase intestine and have limited microbes amounts capable of of hydrolyzing InsP P -inositol phosphates in C). This treatment is thought to enhance the bioavailability ∘ * 5 )are is the ± 6 , reduces myo ) in raw pasteurized activated almonds. At least 24 h of soaking at ambient 6 - InsP 1–6 10 D 11 ). 1 − Prunis dulcis has 12 acid dissoci- 6 content from 14.71 to 14.01 6 into the soaking water. Over a wide pH range, InsP is statistically significant ( 6 6 Low Fe and Zn status has ), also known as phytic acid, In households that rely on 6 2 Almonds ( 3 4–9 Adequateconsumptionofiron(Fe)and 1 Germinating, fermentation, and soaking Phytate, the ionized form of InsP : 117–123 www.soci.org © 2018 Society of Chemical Industry 2 11 99 2019; activated almonds; inositol (InsP); phytic acid; anion exchange chromatography; LC–MS/MS Inositol hexaphosphate (InsP

) via phosphoester bonds (Fig. 1). InsP 1–6 -inositol ring linked with up to six orthophosphate groups

myo- - 20% daily value) of Fe and Zn (both 0.031 g kg D > is ubiquitous inreduces and the is bioavailability considered of an mineral anti-nutrient . that J Sci Food Agric INTRODUCTION Mineral deficiency is anaffects international more public than health one-third of concern the that in world’s population, developing particularly countries. of almonds by degrading nutrient inhibitors, such as phytic acid or BACKGROUND: Activated almonds are raw almonds that have beenfollowed soaked by in a water 24 for 12–24 h drying period h at at low room temperature temperature, (50 sometimes Abstract Lianna Y Lee and Alyson E Mitchell (wileyonlinelibrary.com) DOI 10.1002/jsfa.9151 Determination of Research Article Received: 2 March 2018 Revised: 17 May 2018 Accepted article published: 28 May 2018 Published online in Wiley Online Library: 16 July 2018 a plant-based diet, the lowcessed bioavailability cereals and of Fe contributes to and the Zn prevalencemetabolic of in these unpro- disorders. (Zn) during childhood isgrowth, necessary and to cognitive maintain good development. health, seeds in water, prior toas consumption, potential have strategies all for beenimproving increasing investigated nutrient mineral bioavailability and deficiencies. primary form of phosphatemyo storage in seeds,(InsP and consists of a ation constants ranging, from 1.9 to 9.5,at and physiological is pH. negatively charged spectrometry chromatography coupled with tandem mass ‘activated’ raw almonds using anion-exchange Keywords: decrease from the unsoaked almonds. © 2018 Society of Chemical Industry been correlated with impaired immune function,outcomes, poor pregnancy and increased morbidity. that limits the absorptionhypothesized that of hydrating the essential nutrients during soaking by triggers forming InsP insoluble complexes with minerals such as and zinc. It is temperature was required to reduce InsP CONCLUSIONS: The reduction in InsP of or passive diffusion of InsP binding capacity. RESULTS: Anion-exchange chromatography coupled with tandem mass spectrometry was used to quantify interesting in this regard,( because they are an excellent source di-, tris-, tetra-, penta-, and hexaphosphates (InsP , . - C 2 1 ∘ − 1–5 myo - D cm. All ,InsP mol L concen- 1 : 117–123 μ Ω 6 99 -inositol-1,3,4, were quantified 2019; reduction during myo - 1–6 6 D at longer soak times. Cundervacuumuntil ∘ 90% basis), standards were prepared ≥ 1–3, ) sodium salt (98% assay) 6 ) sodium salt (98% assay), 1 2 ) sodium salt (98% assay), J Sci Food Agric 3 ) were measured because they 24 h). Moisture was determined –InsP reduction. If activation degrades 1 ∼ 6 -Inositol-1,3,4,5,6-pentaphosphate 1–5 were prepared in methanol–water Individual solutions of InsP 6 myo While the effects of soaking and ger- - 21 D ) ammonium salt (98% assay), 15 4 hydrolysis and could improve understanding et al. 6 ,andInsP standards were prepared at concentrations of ) sodium salt hydrate ( 5 6 6 in raw almonds. Levels of InsP -monophosphate (AMP), and pentylamine (PTA) (99% 6 ′ ,InsP -inositol-1-phosphate (InsP 4 myo -inositol-1,5-diphosphate (InsP via enzymatic activity, we would observe an increase in - ) ammonium salt (98% assay), ,InsP 5 D 3 6 3 months) until they were processed. myo Relative to other strategies for dephytinization, soaking is a - < other reagents used were ofcommercial analytical sources. grade and obtained from Samples Raw, propylene oxide (PPO)-pasteurized Nonpareilvested in almonds 2015 har- were obtained from Bluemento Diamond California). Growers Raw, (Sacra- whole almond kernels( were stored at 4 Moisture content analysis The moisture content of almonds wasby determined gravimetrically drying ground samples (1–2 g) at 90 constant weight was achieved ( in triplicate samples. Preparation of reference and analytical standards The preparation ofmethod analytical by standards Duong was adapted from a simple and inexpensivewithout method removing that other canmechanical beneficial processing. remove nutrients phytic acid through heat or EXPERIMENTAL Chemicals and supplies Phytic acid (InsP D present. hydrolysis by-products, especially InsP (5 : 95 v/v) and combinedmethanol–water with the 5 AMP : internal 95 standard v/v). (AMP InsP in assay) were obtained from the(St. Sigma Aldrich Chemical Louis, Company MO, USA). However, if the main mechanism of InsP at concentrations of 1, 5, 10, 20, 30, 40, 50, 60, 80, and 100 5 are products of InsP mination in cerealssimilar and possibilities for legumes tree nuts have remain relativelystudy been unexplored. addresses This widely a lack examined, ofstrategies knowledge concerning for dephytinization edibletion on nuts InsP by evaluating the effect of activa- (InsP were acquired from Alfa Aesar™ via Thermoburg, Fisher PA, USA). Scientific Deionized (Pitts- water was preparedsystem from (Millipore, a Milli-Q Bedford, (MQ) MA) to a resistivity of 18 M activation were by passive diffusion into the water, InsP InsP Additional InsP of the mechanism behind InsP InsP and using liquid chromatography(LC–MS/MS) with tandem and mass anion-exchangesamples soaked spectrometry for chromatography 0, 4, 6, in 8,tinization 10, almond 14, as and a 24 h function toless investigate of dephy- phosphorylation soaking (i.e. duration. InsP Forms of InsP with tration would decrease without increasing the amount of InsP inositol-1,4,5-trisphosphate (InsP 6-tetraphosphate (InsP 8,9 )at 6 www.soci.org LY Lee, AE Mitchell )–which 1 While sev- 15 the effects of © 2018 Society of Chemical Industry Fermentation uses CtolowerthepH. ∘ 5,9,16,17 ,andInsP 7 2 (up to 50%) depending 6 ,InsP 3 These longer soaking periods ).Conformation:5,axial;1,equato- + 20 ,InsP 4 -inositol penta-, tetra-, tri-, di-, and myo The rate of activity depends on -inositol-1,2,3,4,5,6-hexaphosphate (InsP ,InsP 5 degradation by initiating the early stages 4 19 seed germination introduces an additional successively into less phosphorylated inos- 6 myo 6 synthesis and/or activation of enzyme. Phy- C) of 24 h to produce a low-moisture, shelf-stable ∘ Seed variety, maturity, structure, and experimen- In vitro 4 , hydroxyl groups replace phosphate groups. 4,18 1–5 Structure of de novo C; pH 4.5–5.5). ∘ Germination of seeds can also reduce phytate levels by promot- Activated almonds have drawn significant attention as a sim- pH 6–7. Under physiological conditions, the negativeby charges are metal balanced cations, such as sodium (Na rial. In InsP may also induce InsP wileyonlinelibrary.com/jsfa Unlike or fermentation, preparing activatedshorter, requiring almonds 12–24 is h of steepinging followed step by (45–55 a low-heat dry- fermented, and activated almonds.are During steeped sprouting, in water raw (10–12 seeds moist h), rinsed, conditions drained, and (1–3 stored days)mination under at occurs ambient and plant temperature sprouts until appear. ger- Figure 1. of germination and/or optimizing conditions(45–55 for phytase activity seed variety, stage ofture, and germination, pH. moisture content, tempera- incubation period (24–72water h) at after ambient seeds temperature. have been soaked in endogenous bacteria from the seedstion, and to typically acidify requires 2–3 the days soaking at 25–30 solu- have less bindingmineral capacity bioavailability. and a smaller negative impact on tal conditions may impact reported changesresponse in to soaking. phytase The activity impact of in soaking almond kernelsbeen has studied. not ing the activity of endogenousthrough phytase (E.C. 3.1.3.8, and 3.1.3.26) tase hydrolyzes InsP eral studies have shown thatstatistically soaking significant cereals reductions and in InsP legumes yields on the seed variety and soaking conditions, itol phosphates (InsPs) – mono6phosphate (InsP soaking on phytate contentinvestigations remain do inconclusive, not because report other largeto changes soaking. in phytate levels due product. ple method foralmonds, potentially because less enhancing total time the isfermentation. Because required nutritional phytate is than water for value soluble, the sprouting compound or can of be removed by passive diffusion into soaking water.

118 119 1 1 − − C. The mol L ∘ full-scan μ 2 Retention time (min) .MS 1 ) is the concen- − 1 − and 100 nmol g 1 − 100 (1) standard and the AMP L of the 1000 × n Collision recovery was examined using product scan. μ (nmol g u energy (eV) n n a C a C . Total run time was 45 min, wileyonlinelibrary.com/jsfa − 1 C − s 1, 2, 3, 4, 5, and 6) at the begin- C = ) is the concentration of analyte ) . Capillary voltage 4000 V, drying 1 n = z 1 / − L and 200 − Lmin ) μ m μ ( % and AMP ( n Product mass (nmol g C with a flow rate of 10 L min u ∘ C ) is the concentration of analyte measured in ) ratios. Data acquisition and analysis were per- 1 N a − Recovery ) / z L insert (National Scientific, Rockwood, TN). S / μ 259339419499579 79 241659 321 401 481 19 561 19 20 20 8.77 23 10.22 25 13.03 16.37 17.47 22.16 m ( standards solution ( Specifications for multiple reaction monitoring of the par- n Parent mass (nmol g s C is the mass-to-charge ratio. z 1 2 3 4 5 6 C with flow rate 10 L min / ∘ m An Agilent 1260 Infinity LC isocratic pump was used for the Conditions for ESI-MS/MS were as follows: nebulizer pressure InsP InsP InsP InsP InsP AMP 346 79 20 9.41 Analyte ent and product of InsP Table 1. InsP a Retention times displayed for each analyte. ning of sample extraction, such that 50 nmol g where 25 min was used forwashing compound and separation, equilibrating and the 20 column min withbetween for 100% runs. Retention mobile times phase of each B InsP post-column addition of the PTA solution via a mixing teeinner (0.12 mm diameter, 400 mm length;Clara, Agilent CA). Technologies Inc., Column Santa combined temperature flow rate was was maintained 400 at 20 sample was addedaccording respectively. to Association Recovery of Official (%) Analytical Chemists: was determined mixed InsP measured in non-spiked samples, and Analytes were monitored by multiple reaction monitoringmized at collision opti- energies (Table 1). Matrix effects TheinfluenceofalmondmatrixonInsP tration of analyte added to the test sample. in solutions spiked with 100 where the spiked sample, gas temperature 300 mode was used to identify the precursormentation ion and voltage the optimal of frag- eachuct ion(s) analyte. were Collision optimized for energy each and InsP prod- internal standard are summarized in Table 1. Electrospray ionization MS The HPLC instrumentquadrupole tandem was mass spectrometer (Agilent Technologies Inc., coupledSanta to Clara, CA) an with Agilent anESI electrospray source 6460 ionization was (ESI) triple source. operatedsignal-to-noise The ( in the negativeformed with mode MassHunter to software (Version yieldnologies B.06.00, higher Inc.). Agilent All Tech- samples werevial transferred with a into 400 an auto-sampler 55 psi, nozzle voltage300 400 V, sheath gas (nitrogen) temperature 1 1 24 − − and with Cfor ∘ 21 PTA in mol L Lmin C for up 1 ∘ μ μ ,24min, et al. − flow rate) g et al. 1 × − C. The residue ∘ L sample solution Lmin μ 25 μ in a convection oven at et al. 1 C. Samples were dried to − . A mixed standard with ∘ 1 -inositol phosphates www.soci.org 2 − acetic acid for 3 h (300 rpm, AMP (925 ± 1 myo 1 − - sodium hydroxide (NaOH) and − D mol L 1 μ forms were eluted at 200 − n mol L μ was prepared and combined with AMP. AMP). Duplicates of 0.5 g almond powder were 1 ethylenediaminetetraacetic acid disodium m) from Thermo Scientific (Pittsburg, PA). : 117–123 © 2018 Society of Chemical Industry 1–6 − μ acetic acid. The solution was transferred to a 23 1 C for 13 h. The solution was cooled to ambient 99 C until used. 1 − ∘ ∘ − 20 C until analyzed. of InsP aqueous ammonium carbonate, pH 9.0 (solvent A), 2019; ∘ − L, and the InsP 1 , the eluents were mixed with 30 mmol L 1 μ − 1 − 20 − 2.1 mm, 3 , 20 min). The final solution was filtered through a − EDTA) in 0.75 mol L × L of 1 mmol L Briefly, 0.18 g of phytic acid sodium salt was dissolved in 2 g C for 24 h. Dried almonds were ground and sifted through μ forms were separated with an Agilent Infinity 1290 ∘ mol L × m Millex™ nylon syringe filter (EMD Millipore, Pittsburg, PA, 22 Lmin μ 5 μ n μ C). To prevent formation of insoluble complexes between C), vortex mixed (60 s), and centrifuged (3005 ± ∘ ∘ The reference standard solution containing all six InsPs was Sample preparation was adapted from a method by Liu and 75 high-performance LCusing (HPLC) a system100 weak mm (Wilmington, anion-exchange DE, column USA) (Hypersil GOLD AX, glass vial, flushed with nitrogen, sealed, and heated at 125 final moisture content of 3.40–4.06 g kg 3 h, and then at 70 temperature before it was evaporatedVac System to (Savant, dryness Holbrook, using NY,was a USA) reconstituted Speed set in to 2.5 40 mLand methanol–water stored at solution (5 : 95 v/v) Sample preparation To prepare control andkernels activated were soaked in almonds, 500 mL 100 of g Milli-Qfor water of 0, in triplicate 4, raw, batches 6, whole 8, 10, 16, and 24 h at 24 and methanol in water, 5 :size 95 was v/v (solvent 10 B). The injected sample 1000 J Sci Food Agric using a 20 min4 gradient min, program followed beginning byover with 0–20% 14 solvent min. A B The overB column for 2 min, was for washed and 20 with finally min400 100% 20–55% at mobile the A phase end of each run. For a total flow rate of The mobile phases200 mmol for L the chromatographic separation were to 1 week between sample injections. prepared using a modified procedure based on Duong in each solution. Linear regression was usedcurves to for construct each standard InsP. Alltions standards at were the beginning prepared of from sample injection stock and solu- stored at 4 Impact of almond activation on 150, 200, 300, 350, and 400 salt (Na 50 a 35-mesh (0.417 mm opening)Mentor, OH) Tyler to standard ensure a screen uniform powder (W.S. size.stored Tyler, Almond powder at was some modifications. 0.22 vortex mixed for 60 s.twice To with remove 3 fats, mL the aliquots solution ofThe was hexane extracted hexane and vortex layer mixing for(3005 was 1.5 min removed each time after centrifugation USA) and combined withfinal the concentration AMP of standard solution 75 to attain a 60 mL of 3.2 mol L The final concentration of the internal standard was 75 extracted with 3 mL of 3.2 mol L LC–MS/MS analysis InsP minerals and phytate, 12mL mL of 0.11molL of supernatant was combined with 20 24 Chen. in a post-columnaccording reactor to the method to by Rougemont reduce sodium adduct formation a 5 : 95 v/v methanol–water solution (200 . ) 5 6 1 4 n 6 1 26 − − con- 26 3 analysis Though 0.001). : 117–123 EDTA can can form and InsP 2 < 16 1–6 n 99 4 P levels had to will likely be isolate, 0.11% 6 Lower recovery ,andInsP 1 6 was spiked into 2 − content for most 2019; concentration by 23 content decreases 6 InsP for InsP 6 1–6 6 EDTA in 0.75 mol L Cresultedinaminor 0.05) from the con- ,InsP 2 by 7–12%. ∘ 1 InsP concentrations in raw, > 6 et al. Na Addition of Na P 2,9,26,27 levels increased after 6 h 1 1–6 − demonstrating satisfactory 21 4,9,16 5 C. 23 ∘ 0.05). InsP J Sci Food Agric < in the mobile phase. Polyamines 0.05) increases by 38% and 35% P 1 < and InsP L of in-house reference standard per − μ P 4 and soaking pea cultivars for 6–18 h sample before a significant increase of 9 1 reduction after soaking various cereals and − had to be below 0.01 mg g 6 6 20%), < has up to 12 ionizable protons, pH plays a crit- compounds, and this is more apparent at lower 0.05), and concentrations continued to increase n n < In order to prevent precipitation and loss of InsP 0.05) between different soaking times. The amount extraction methods (68% to 111%). P 25 > 21 n P 0.05) (Table 4). After 24 h of soaking, InsP in unprocessed, raw almonds determined in this study n > reduction ( , the reference standard containing InsP P 6 n To evaluate the matrix effects of almond meal on recovery of the Because InsP The effect of soaking time on InsP Our observations are in agreement with previous findings that gram dry sample) corresponding to recovery(Table 1 3). and Recoveries 2 ranged respectively fromcompares with 73% the recoveries determined to by previous 101% studies with similar (Table InsP 3), which is comparable to results reported by Liu InsP pasteurized almonds is summarizedthat in activating almonds Table 4. for Results 24 h indicate reduces InsP is a potent inhibitor even at low levels. The amount of InsP demonstrated significant ( Steeping in water forInsP 24 h at 25 over three consecutive days2.59% and and % 4.92%. RSD values ranged between during sample preparation, 0.11 mol L NaOH was mixed with sample extract (2was : 1 defatted v/v) before using the hexane. solution Intively charged addition, phosphates the to affinity cationsreduced of in the the signal nega- sample (e.g. sensitivity sodium) and suppressed ionization of InsP ical role in the recovery of InsP. At a low pH, InsP by 0.21–2.00% betweenference 4 is and not 14trol h statistically sample of significant (Table activation, 4). ( but InsP the dif- and seeds remained between 86% and 100% after soaking. insufficient for influencing mineral bioavailability, because InsP centrations remain constant, as mean valuesdifferent were ( not statistically using LC–(ESI-)MS/MS) in almonds, at ambient temperature decreased InsP of soaking ( legumes in water for 6–24 h at 25 such as PTA remove sodium adductsMS analysis. by creating ion pairs during percentages suggest that the almondization matrix of interferes InsP with ion- 4.75% from the initial value ( be less than 0.3 mg g concentration levels. of the initial, before Fe absorption increased four- to fivefold. 4.17% in Fe absorption was observed for soy flour ( insoluble complexes with proteinsfrom and solution. To cations, avoid and this4.5–5 precipitate effect, following pH acetic acid can extraction. be adjusted to above extraction. The recovery analysistration was levels conducted (200 at and two 400 concen- extraction of target analytes. report a minimal InsP statistically significant, these reductions in InsP This was resolved with theconcentration post-column addition of of PTA 15 mmol at L a final reduce formation of insoluble saltsfor by cations. acting as strong chelators almond samples at the beginning of the acetic acid (3.2 mol L respectively from the control (Table 4). InsP of InsP between 6 andent 14 ( h, but values were not statistically differ- , 1 n n 3 − (2) )per ,and is the 5 n d ,InsP mol L and the 2 and the . m μ www.soci.org LY Lee, AE Mitchell n n 1–6 ,InsP standard at 4 ,InsP 1 standards and n © 2018 Society of Chemical Industry n concentration of ,InsP 3 1 . − 6 for InsP Herein, InsP of 3 : 1) was 1 pmol for 1 − ,InsP 2 ) is the concentration of N mol L / f 1 S − μ d for InsP 21,23,24 is the total volume of extract × mol L 1 0.99) across the concentration d ,InsP v − μ v 1 > m × (mol L 2 ratio of 3 : 1 and 10 : 1 respectively. R . Intra-day precision for analysis of M in the extract. The amount of InsP M mol L N / μ n 4–6 . The LOQ (S/N of 10 : 1) was 3 pmol for = S 6 C Tukey’s honest significant difference test concentration of each InsP standards ranged between 1.49% and 2.89%. 1 ) is the concentration of analyte (InsP ) in the extract, − 1 n − 1–6 post hoc 3) is the dilution factor to account for the addition , and 1–400 5 = )ofeachInsP ( mol L 1 . f InsP − were calculated from standard solutions using the μ 1 mol g standard over three consecutive days. Precision was d − 1 μ n n ( − and 5 pmol for InsP and 3 pmol for InsP C mol L 0.05. Analyses were conducted in Microsoft Excel, Version in almond samples (micromoles of analyte per gram dry μ mol L ,andInsP < 1–5 1–3 6 4 mol L μ μ P Intra-day variability of the method was evaluated by analyz- evaluated by calculatingAccuracy was relative evaluated by standard calculating the ratiotal deviation of to the experimen- (% the nominal RSD). concentration100 of each standard at 10, 50, and wileyonlinelibrary.com/jsfa 14.4.3 (Microsoft, Redmond, WA). at InsPs are negatively chargedby anion-exchange compounds chromatography, suited and forionized they by are negative-ion separation electrospray. preferentially RESULTS AND DISCUSSION was evaluated byfollowed a by the one-way factorial analysis of variance and per dry mass of almonds were calculated using: Method validation The analytical method was evaluatedaccuracy. The for calibration linearity, curves precision, were determined and inwere triplicate linear and over a range of 1–100 Inter-day variability of each standard was measured at 80 InsP The calibration curves constructed using the InsP minimal accepted value of The effect of soaking time on the average amount of each InsP three times on the samewas day at assessed 7 h by intervals. Inter-day measuring variability the 50 where internal standard AMP eluted within 25 min. Each InsP of 2 mL ethylenediaminetetraacetic1 mL acid–NaOH(aq) sample supernatant after solution acetic acid extraction, to and Quantitation of InsP analytes in almond samples To determine the amount of InsP AMP eluted in the sameand order almond in samples (Fig. commercial 2(B)), standards but (Fig. retention 2(A)) in times of sample the matrix analytes were slightly delayed.in Peak samples, shapes possibly were due broader to the higher abundance of InsP (0.003 mL), mass), a standard calibration curve waslyte. constructed The for regression each model ana- was usedtion to ( determine the concentra- 80 each InsP AMP achieved strong linearity ( InsP Statistical analysis The limit of detection (LOD)each and InsP limit of quantification (LOQ) for mass of almond meal corrected for dry weight. ing the 50 gram dry mass of almonds, each analyte (InsP ranges of interest (Table 2). The LOD ( InsP InsP

120 121 6 Cere- 4

based on the endoge- 6 (A) (B) wileyonlinelibrary.com/jsfa in soaked seeds, which reported 6 This information corresponds with 28 ) LOD (pmol) LOQ (pmol) 1 − , identical soaking conditions lowered InsP mol L which suggests that different seed types have μ reductions in soaked cereals (e.g. rice, rye, and trit- standard from three replicate analyses et al. 5 n 6 Linear range ( matrix composition specific to different plantby varieties. Lestienne In a study 20–30% InsP icale) compared with 0–14% dephytinization in soaked legumes concentration in some cerealsrice and and ) legumes but (e.g.mung not millet, ), others , (e.g. sorghum, , and another study comparing InsP distinctive capacities to hydrolyze InsP nous phytase activity and/or the seed matrix interactions. als and pseudo-cereals tendactivities, to while exhibit legumes highphytate-degrading phytate-degrading and activity. oilseeds exhibit a tenfold lower -monophosphate (AMP) detected in (A) commercial standards and (B) almond 16 ′ 2 R content 6 InsP ) 9 2 and adenosine 5 after soaking dif- 10 For example, soak- × 6 reduction observed 1–6 6 by 54% were reported 9,16,17 5 Our findings differ due to -inositol phosphates www.soci.org 17 myo ) as a result of soaking in water -

D

and InsP 1 2 3 5 6 4 6 1 2 3 5 4 6 )Intercept( InsP InsP InsP AMP InsP InsP InsP InsP InsP AMP InsP InsP InsP InsP 2 C led to 42–59% removal. 10 ∘ × : 117–123 © 2018 Society of Chemical Industry 0.0150.0120.0150.0150.004 0.1301 0.1308 0.1497 0.1171 0.1103 0.996 0.996 0.993 0.999 0.998 1–100 1–100 1–100 1–100 1–400 1 1 1 1 3 3 3 5 5 5 0.027 0.1349 0.998 1–100 1 3 99 Phaseolus vulgaris 2019; hydrolysis by endogenous phytase. Linear range, correlation coefficient, slope, intercept, LOD, and LOQ for each InsP 6 LC–(ESI-)MS/MS chromatograms of InsP C) at different pH (4.5–8.0). ∘ 2 3 4 5 6 1 Another explanation for the limited InsP Studies that report large reductions in InsP Analyte Slope ( InsP InsP InsP InsP InsP InsP Table 2. powder samples. in black beans ( (50–60 J Sci Food Agric Significant reductions in InsP Figure 2. Impact of almond activation on dissimilarities in soaking conditions,rate which of InsP would influence the in pea seeds decreased 67–83% after 48 h soaking or sprouting. ing preheated rice at 10 in our study may be differences in phytase activity and seed ferent cereals and legumes usuallytreatments, combined such soaking with as other preheating thetemperature of rice, soaking, increasing or the adjusting time pH. and 6 loss (%) : 117–123 6 (%) b 99 InsP 2019; Recovery after 0–24 h of soaking compounds with fewer 6 is less apparent, as the 6 0.37a0.32a 0 0.21 0.18a0.17a 0.96 1.47 0.49a 1.28 0.36a 2.00 0.56b 4.75 1–3 ± ± ± ± ± ± ± 1–3 InsP J Sci Food Agric , after 24 h of soaking (Table 4), lead to limited improvements in 5 5 were measured in our study to 1–5 and InsP and InsP 4 5 )1 2 4 0.19c 14.01 0.16a0.02a 14.71 14.74 0.38b0.10b 14.57 14.49 0.31b 14.52 1 0.25bc 14.42 hydrolysis to InsP − ± ± ± ± ± ± ± InsP 4–6 The change in InsP mol g ), and their quantity may fall below the LOQ as 1 μ − 28,34 0.020.02 85.7 94.3 0.040.050.030.020.08 73.5 78.8 93.2 95.2 76.4 97.5 88.3 100.9 93.5 98.0 0.39 0.10 0.08 0.26 0.02 ± ± ± ± ± ± ± ± ± ± ± ± hydrolysis over the 24 h soaking period, but the corre- and recovery (%) of analytes in 14 h activated almond samples a mol g 6 μ 0.05). 4 0.16c 4.82 0.14a0.03a 3.54 3.72 0.35b 4.15 0.20b 4.22 0.14b 4.46 > 0.13ab 4.15 L of reference standard per gram almonds. 5.4 Concentrations of InsP P ± ± ± μ ± ± ± < ± InsP sample) these are not availableand for are commercial not the consumption form in of almond the that people USA are ‘activating’. sponding rises in InsP mineral bioavailability. Additional treatments would beto necessary continue InsP CONCLUSION The results of this study suggestthe that activation does nutritional not enhance valuecantly of reducing phytic raw acid pasteurized levels. almonds At by least signifi- 24 h of activation by phytase. negative charges and weaker binding capacity. may be attributed to stepwise release of phosphate from InsP they diffuse into thein soaking InsP water. Phytase was likely involved intrinsic concentration of these( compounds in the seed is low examine the rolealmonds. Levels of of InsP phytase in dephytinization in activated 1 − 1 − or 6 mol g The μ 29 www.soci.org LY Lee, AE Mitchell 3 0.06a0.04a0.20a 2.34 2.44 2.79 0.09a0.15a 2.71 2.89 0.13a 2.84 0.09a 3.27 0.62 g kg ± ± ± ± ± ± ± ± InsP © 2018 Society of Chemical Industry ) Amount detected ( 1 − C) for almond activation ∘ 1–6 5 (micromoles analyte per gram dry mass almonds) and percentage loss of InsP However, temperatures ± mol g μ a,b 2 31 0.08a0.03a0.21a 2.22 2.35 2.51 0.06a0.17a 2.39 2.43 0.11a 2.34 0.07a 2.39 With respect to seed matri- 4 ± ± ± ± ± ± ± C) and steam pasteurization InsP ∘ 1.251.760.411.430.735.56 2.32 4.34 3.06 3.91 3.69 14.70 2.012.820.632.291.178.90 3.68 6.79 4.00 5.89 5.24 15.95 reduction after soaking may be 6 C threshold at which phytase begins ∘ 1 0.03a0.01a0.07a 1.87 2.15 2.21 0.02a0.05a 2.09 2.19 0.03a 2.10 0.02a 2.11 ± ± ± ± ± ± ± C followed by drying (50 L of reference standard per gram almonds; recovery 2: 400 content in raw almonds ∘ μ hydrolysis, InsP 2 form, mean plus/minus standard deviation followed by the same letter is not significantly different across different cultivars according If heat exposure were a significant factor that 6 may interfere with the passive diffusion of InsP 1–6 n degradation via thermal inactivation of phytase. ± 33 6 The effect of almond matrix on recovery. Level of InsPs ( InsP 29,30 can surpass the 60 32 5 6 1 2 3 4 C) C, unpasteurized almonds would need to be used. However, ∘ The mass of almonds with different soaking times has been adjusted for dry mass using the moisture content (percentage dry basis) of each Recovery 1: 200 For each InsP The mass of almonds has been adjusted for dry mass using moisture content (percetage dry basis). ∘ The use of pasteurized almond samples may also influence the Time (h) InsP InsP InsP InsP InsP InsP spiked with in-house reference containing InsP to one-way analysis of variance and Tukey’s honest significant difference test ( in water at 24 a b 40.69 60.70 00.66 Table 4. b a AnalyteInsP Amount added ( Table 3. corresponding treatment group. Values represent mean plus/minus standard deviation of six replicates. 80.66 10 0.66 14 0.63 24 0.66 to denature. more pronounced in unpasteurized almonds.effects of To activation understand without prior the exposure to temperatures60 above wileyonlinelibrary.com/jsfa by weight) (e.g. , , and ). (95 during PPO treatment (up to 71 prevented InsP Pasteurized almonds are consideredstudy, raw because and previous were studies used havenor for indicated that this fumigant neither heat pasteurizationand diminished sensory the attributes nutritional of value almonds. activation of by impeding water migration. rate of InsP high fat content in almond seed cotyledons (43.36 ces, phytase and other enzymeslocated needed in during germination the are pounds, embryo, consisting of while proteins, metabolized lipids,contained and in food the starches, cotyledons storage are surrounding primarily the com- seed embryo.

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