Quantitation of Three Strecker Aldehydes from Enzymatic Hydrolyzed

Sucrose, glucose, and fructose were purchased from Ajax Finechem (NSW, ... After that, the fiber was exposed in the headspace of the sample and the in...
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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Quantitation of Three Strecker Aldehydes from Enzymatic Hydrolyzed Rice Bran Protein Concentrates as Prepared by Various Conditions Supeeraya Arsa,† Chockchai Theerakulkait,*,‡ and Keith R. Cadwallader§ †

Faculty of Agro-Industry, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand Department of Food Science and Technology, Kasetsart University, Bangkok 10900, Thailand § Department of Food Science and Human Nutrition, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States

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‡

ABSTRACT: The quantitation of three Strecker aldehydes2-methypropanal (2-MP), 2-methylbutanal (2-MB), and 3methylbutanal (3-MB)from rice bran protein hydrolysate (RBPH) prepared under various conditions were investigated. The preparation conditions included hydrolysis time (0, 0.25, 2, 4, or 8 h), pH adjustment (pH 4.0, 7.0, or 10.0), and sugar addition (sucrose, glucose, or fructose). These conditions provide a significant potential for aroma generation from the Strecker degradation and Maillard reaction. The Strecker aldehyde quantitation was performed using gas chromatography (GC) with cryo-focusing technique. These combined techniques encourage the precise 2-MB and 3-MB quantitation. The highest concentrations of three Strecker aldehydes were found in RBPH that was prepared by alcalase hydrolysis at 4 h with fructose addition (RBPH-F) and adjusted to pH 7.0 before spray drying. Thirty-nine aroma-active compounds of RBPH-F were discovered using solid-phase microextraction coupled with GC-olfactometry. KEYWORDS: enzymatic hydrolysis, gas chromatography, cryo-focusing technique, rice bran protein, Strecker aldehydes

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INTRODUCTION Rice bran protein is an interesting, high-quality, low-price, and natural protein source with good biological values and digestibility.1,2 However, rice bran protein is not commercially useful because of its unpleasant characteristic odor.3 Enzymatic hydrolysis of rice bran protein can produce the flavor substrates as free amino acids or smaller peptides. These hydrolysis products can react with reducing sugars in the Maillard reaction to form numerous aroma compounds. Our previous studies showed that rice bran odor could be improved by enzymatic hydrolysis4 and a spray-drying process.5 The aroma attributes of rice bran protein hydrolysate (RBPH) were dominated by preparation conditions.4,5 The desirable aroma attributes such as cereal-like, nut-like, milk-powder-like, sweet, and cocoa-like aromas were found in RBPH powder prepared by alcalase hydrolyzed with fructose addition before spray drying. Additionally, there were nine odor-active compounds in RBPH and the highest odor-active values were Strecker aldehydes.6 Therefore, rice bran protein has the potential to be used as a precursor for Strecker aldehyde formation. The Strecker aldehydes, 2-methypropanal (2-MP), 2methylbutanal (2-MB), and 3-methylbutanal (3-MB), are a few volatiles found at parts per million threshold levels. These volatile compounds are important contributors to the malty aroma characteristic.7,8 Additionally, they are also identified as having chocolate aroma attributes from gas chromatographyolfactometry (GCO).9,10 The three Strecker aldehydes are lowmolecular-weight compounds and easy to volatilize. In addition, 2-MB and 3-MB are relevant isomers and they have the same molecular weight and similar structure which causes difficulty separating them.11,12 2-MB and 3-MB cannot © XXXX American Chemical Society

be separated by using atmospheric pressure chemical ionization-time-of-flight mass spectrometry.13 The use of stable isotope dilution assay (SIDA), [2H2]-2-methylbutanal and [2H2]-3-methylbutanal, has been reported in the isolation of 2-MB and 3-MB of cocoa powder.7 Although SIDA is a very accurate quantitation method, the expense of the isotopic analogues synthesis is high.14 Consequently, gas chromatography (GC) with cryo-focusing technique was utilized and combined with SIDA method for 2-MB and 3-MB separation and for precise quantitation in this study. The Maillard reaction is known to be a crucial reaction for aroma generation. Most aroma-active compounds, which are the principle aroma profile of cocoa and chocolate, are derived from the Maillard reaction.14 The intermediate and final stages of the Maillard reaction are the most important to flavor development, especially the so-called Strecker degradation step, in which amino acids react with dicarbonyls formed previously in the reaction, leading to the amino acids deamination and decarboxylation.15 The three Strecker aldehydes are predominantly formed by Strecker degradation of the amino acids such as valine, isoleucine, and leucine, resulting in their respective aldehydes 2-MP, 2-MB, and 3MB.16,17 The abundance of isoleucine and leucine in acidhydrolyzed18 and enzymatic-hydrolyzed rice bran protein6 encourage the use of rice bran protein for the three Strecker aldehyde formations. Received: Revised: Accepted: Published: A

April 1, 2019 June 24, 2019 June 28, 2019 June 28, 2019 DOI: 10.1021/acs.jafc.9b02025 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Effect of pH Adjustment before Spray Drying. The 5% protein solution of RBP was prepared as described above and hydrolyzed by alcalase at 55 °C for 4 h. The alcalase was inactivated. Then fructose (0.1 M) was added into the liquid RBPH, and the liquid RBPH was adjusted to pH 4.0, 7.0, or 10.0 with 1.0 mol/L HCl or 1.0 mol/L NaOH before heating to 95 °C for 10 min. After that, the suspension of liquid RBPH was dried using a spray dryer. The resulting products after spray drying were RBPH-F with different pH adjustments. Then the concentrations of Strecker aldehydes in RBPHF were evaluated. Effect of Sugar Type. The 5% protein solution of RBP was prepared as described above and hydrolyzed by alcalase at 55 °C for 4 h. The alcalase was inactivated. Next, 0.1 M sucrose, glucose, or fructose was added into the liquid RBPH before adjusting to pH 7.0. Then the liquid RBPH was heated to 95 °C for 10 min and spraydried. The resulting product was the hydrolysate powder with different sugars added. Finally, the concentrations of Strecker aldehydes and aroma-linking score of the hydrolysate powder with different sugar additions were evaluated. Aroma Characteristic and GC-MS Analysis. RBPH-F, which was prepared by RBP hydrolysate for 4 h with fructose added and adjusted to pH 7.0 before spray drying, was determined by using the headspace solid-phase microextraction (SPME). The SPME fiber was coated with a divinylbenzene/carboxene/polydimethylsiloxane 50/30 μm (Supelco Inc., Bellefonte, PA, USA). The aroma characteristics of RBPH-F were evaluated by using SPME-GCO technique. One gram of RBPH-F was dissolved in 5 mL of saturated NaCl in 20 mL glass vials. The sample vials were sealed with a silver aluminum seal pressure release (open center) with a 20 mm × 2.5 mm × 5 mil PTFE/silicone liner. Then the sample vials were equilibrated at 60 °C for 15 min. After that, the fiber was exposed in the headspace of the sample and the inlet for 30 and 20 min, respectively. GCO analysis was performed using a 6890 GC (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and a sniffing port (DATU, Geneva, NY, USA). Helium gas was used as carrier gas at a constant flow rate of 2 mL/min. The GC oven was programmed at 40 °C, held for 5 min, then heated to 225 °C at a rate of 10 °C/min, and held for 40 min on a polar column (RTX-wax, 15 m × 0.32 mm i.d. × 0.5 μm film thickness; Restek, Bellefonte, PA, USA), whereas a rate of 6 °C/min was set for a nonpolar column (DB-5, 15 m × 0.32 mm i.d. × 0.5 μm film thickness; Restek). The inlet temperature was set at 260 °C with spitless injection (purge flow 50.0 mL/min for 4 min). Four experienced panelists (1 male and 3 females) sniffed and evaluated the odor description and aroma intensity. The odor description was listed when the odor was detected by 3 out of 4 panelists. After that, retention indices (RI) of odor description from GCO were matched and their RI was confirmed with the compound from GC-MS. RI’s were obtained by using the retention times of n-alkanes (C5−26 for DB-5 and C5−30 for RTX-wax) under identical conditions for GCO and GC-MS. The aroma compound identifications were performed by matching the RI of different polar column phases with the literature, comparing mass spectra with NIST 2.0d library, reference standards, and odor description. For GC-MS analysis, Stabiwax (30 m × 0.25 mm i.d.; 0.25 μm film thickness; Restek) and SPME were conducted using the same method as with GCO as previously described. Ten microliters of internal standards of methanol (butanal (1.02 mg/mL), 2-methyl-3-heptanone (0.08 mg/mL), 6-undecanone (0.073 mg/mL), and 2-ethylpyridine (0.106 mg/mL)) were spiked into the sample vials before they were equilibrated. After that, the fiber was desorbed for 20 min in a 6890 GC (Agilent Technologies Inc., Palo Alto, CA, USA) equipped with a 5973N mass selective detector (Agilent Technologies Inc., Palo Alto, CA, USA). The flow rate of helium gas was 0.8 mL/min. The GC oven was programmed at 35 °C, held for 5 min, then heated to 225 °C at a rate of 4 °C/min, and held for 30 min. The inlet temperature was set at 260 °C with spitless injection (purge flow 50.0 mL/min for 4 min). The ion source temperature was at 280 °C, mass spectra were obtained by electron impact ionization at 70 eV, and data were

However, some preparation conditions, such as hydrolysis time, sugar, and pH adjustment before spray drying, that need to be assessed as quantitative yields of the three Strecker aldehydes in RBPH have not been reported yet. These preparation conditions are especially crucial parameters that affect peptide size, free amino acid concentrations, and the three Strecker aldehydes generated from the Strecker degradation and Maillard reaction. Therefore, the purpose of this study was to identify the aroma-active compounds of RBPH. Moreover, the effect of preparation conditions (hydrolysis time, pH adjustment, and sugar-type addition before spray drying) on the formation of the three Strecker aldehydes; 2-MP, 2-MB, and 3-MB of RBPH was determined. Additionally, the three Strecker aldehydes were quantitated by using SIDA method combined with GC with cryo-focusing technique.

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MATERIAL AND METHODS

Materials. Rice bran from jasmine rice (Oryza sativa L., Variety Khao Dawk Mali 105) obtained from Patum Rice Mill and Granary Company, Ltd. (Bangkok, Thailand). Chemicals. Alcalase 2.4 L FG (EC 3.4.21.62, proteinase from Bacillus licheniformis) was obtained from Novo Co. (Novo Nordisk, Bagsvaerd, Denmark). Papain (EC 3.4.22.2, proteinase isolated from papaya latex (Carica papaya) 3.11 U/mg) was purchased from Fluka (Fluka BioChemika, Buchs, Switzerland). All chemical reagents were analytical grade; HCl and NaOH came from Merck (Darmstadt, Germany). Sucrose, glucose, and fructose were purchased from Ajax Finechem (NSW, Australia). NaCl and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). The reference standards of 2-MP, 2-MB, 3-MB, hexanal, (Z)-4-heptenal, octanal, 2,5dimethylpyrazine, dimethyl trisulfide, trimethyl pyrazine, 2-ethyl-5methylpyrazine, 2,5-diethylpyrazine, furfural, methional, (E)-2-nonenal, phenylacetaldehyde, 4-vinyl-2-methoxyphenol, and 4-hydroxy-3methoxybenzaldehyde were obtained from Sigma-Aldrich Co. (St. Louis, USA). [2H2]-3-methylbutanal was purchased from CDN isotopes (Quebec, Canada). Preparation of Defatted Rice Bran and Rice Bran Protein Concentrate (RBP). Defatted rice bran and RBP were prepared by following the method of Arsa and Theerakulkait.6 Rice bran was defatted twice using hexane, followed by air drying overnight in a fume hood. Then defatted rice bran was dispersed in distilled water at a ratio of 1:4 (w/v) and adjusted to pH 9.5 with NaOH. Then the suspension was centrifuged and the supernatant was adjusted to pH 4.5 with HCl followed by centrifugation. The precipitate was RBP. Effect of Enzymatic Hydrolysis. The 5% protein solution of RBP was prepared by dispersing RBP in distilled water. The protein solution was adjusted to pH 8.0 with 1.0 mol/L NaOH. Then papain or alcalase was used at the same concentration of 1.25 AU/g rice bran protein and at 37 and 55 °C for 0, 0.25, 2, 4, and 8 h, respectively.6,19 The papain or alcalase in liquid RBPH was inactivated by heating in boiling water for 15 min. The liquid RBPH was evaluated for the degree of hydrolysis (DH), which was calculated as follows20 %DH =

soluble nitrogen in trichloroacetic acid supernatant (mg) × 100 total nitrogen in sample (mg)

Effect of Hydrolysis Time. The 5% protein solution of RBP was prepared as previously described and hydrolyzed by alcalase at 55 °C for 0, 0.25, 2, 4, and 8 h.6,19 After that, alcalase was inactivated by heating in boiling water for 15 min. Then fructose 0.1 M was added into each liquid RBPH before adjusting to pH 7.0. The liquid RBPH was heated to 95 °C for 10 min and spray-dried by using a spray dryer (Basic model, GEA Niro A/S, Thailand; inlet 160 °C, outlet 100 °C). The resulting product was RBPH with fructose added and spray-dried powder (RBPH-F) at different hydrolysis times. The amounts of three Strecker aldehydes and aroma-linking score of RBPH-F were determined. B

DOI: 10.1021/acs.jafc.9b02025 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry acquired across the range m/z 35−350 amu in scanning mode with a rate of 5.27 scan/s. Strecker Aldehyde Quantitation. Special techniques such as SIDA and GC with cryo-focusing technique were applied for Strecker aldehyde quantitation. A half gram of the RBPH-F was dissolved in 5 mL of saturated NaCl in 20 mL glass vials. Ten microliters of [2H2]-3methylbutanal (4 μg/μL in methanol) was spiked as an internal standard into sample vials. The sample vials were sealed and equilibrated to 60 °C for 15 min. After that, the fiber was exposed in the headspace of the sample for only 5 min. The Strecker aldehydes concentrations were analyzed with a 6890 GC-5973N mass selective detector in the cool-splitless mode by using a Gerstel CIS4 injector (Gerstel GmbH & Co. KG, Germany) (−50 °C initial temperature, ramped at 12 °C/s to 260 °C with a final hold time of 20 min). A SAC-5 column (30 m × 0.25 mm i.d.; 0.25 μm film thickness; Supelco Inc., Bellefonte, PA, USA) was used to separate the volatiles. The GC oven temperature was programmed from 10 to 250 °C at 8 °C/min with initial and final hold times of 5 and 30 min, respectively. Helium was used as carrier gas at a constant flow rate of 1.3 mL/min. The capillary direct interface temperature was 280 °C. The mass spectrometric data analysis was carried out using continuous scanning mode from m/z 35−350 amu with a scan rate of 5.27 scan/s. The electron ionization mode was 70 eV. The concentrations of Strecker aldehydes were calculated using the following equation:

comparison of retention index, mass spectra, and odor description with reference standards were conducted to identify the compounds of RBPH-F. According to the GCO and GC-MS results, odor descriptions and compounds in RBPH-F were malty (2-MP, 2-MB, and 3-MB), boiled potato (3-(methylthio)propanal), rosy or floral (phenylacetaldehyde), and nut, roast, or coffee (pyrazines: trimethyl pyrazine, 2-ethyl-5-methylpyrazine, 3ethyl-2,5-dimethylpyrazine, and 2,5-diethylpyrazine). These compounds were derived from the Strecker degradation and the Maillard reaction. The Strecker degradation of valine, isoleucine leucine, methionine, and phenylalanine resulted in their respective aldehydes 2-MP, 2-MB and 3-MB, 3(methylthio)propanal, and phenylacetaldehyde.16,17,21 Heat treatment and sugar addition primarily influenced pyrazine formation; therefore, pyrazines might be derived from both the Strecker degradation and the Maillard reaction.22 The burnt sugar or caramel-like odor (4-hydroxyl-2,5-dimethyl-3(2H)furanone) were also derived from the Maillard reaction.18 The conversion of hexoses, such as fructose or glucose, created 4hydroxy-2,5-dimethyl-3(2H)-furanone.23 Aldehydes were responsible for fresh, green, or fatty odor. Hexanal, (Z)-4-heptenal, octanal, (Z)- and (E)-2-nonenal, (E,Z)-2,6-nonadienal, (E)-2-decanal, (E,E)-2,4-nonadienal, and (E,E)-2,4-decadienal were presented in RBPH-F. Most of the aldehydes were derived from lipid oxidation.24 Hexanal and (E,E)-2,4-decadienal were obtained from the oxidation of linoleic acid, while octanal and nonanal are derivatives of oleic acid.25 There were some compounds derived from phenolic compounds or lignin degradation18 such as 2-methoxyphenol (smoky), 4-methylphenol (dung or stable), 4-vinyl-2-methoxyphenol (spice or clove), and 4-hydroxy-3-methoxybenzaldehyde or vanillin (vanilla). These aroma compounds were also detected in acid-hydrolyzed RBP.18 Vanillin originated from lignin degradation in the presence of air and it can be formed by thermal degradation of ferulic acid.26 4-Vinyl-2-methoxyphenol was obtained from thermal decarboxylation of ferulic acid,18 which was the most abundant phenolic acid in rice bran (data not shown). As a result of the various types of volatile compounds in RBPH-F, the three Strecker aldehydes, 2-MP, 2-MB, and 3MB, were of great interest. Because of the high amounts of precursors, free amino acids such as leucine, valine, and isoleucine were elucidated in liquid RBPH.6 Therefore, the Strecker aldehydes; 2-MP, 2-MB and 3-MB, in RBPH-F were evaluated in different preparation conditions. However, the separation of 2-MB and 3-MB by human nose was difficult. 2MP and 3-MP could not be completely separated by polar column because they coeluted on polar column.27 Therefore, special techniques were needed for accurate 2-MP and 3-MP quantitation as mentioned above. The SIDA method combined with GC with cryo-focusing technique was used in this study. Effect of Enzymatic Hydrolysis. The degree of hydrolysis of liquid RBPH with no enzyme and with alcalase or papain hydrolysis is shown in Figure 1. The alcalase hydrolysis had significantly higher DH than hydrolysis by papain and no enzyme conditions at every hydrolysis time (p ≤ 0.05). As a result of their specificity, the types of polypeptide fragments released in the hydrolysate differ between proteases.28 Alcalases are the most commonly used enzymes and it is an endoprotease which attacks peptide bonds in the interior of the polypeptide chain.28 Many publications report the higher DH of alcalase hydrolysate than those of other enzymes. The

concentration(μg/g of sample) =

mass of labeled (μg) area of unlabeled × × Rf area of labeled mass of sample (g)

Mass ion of unlabeled (standard) compounds, labeled compounds (isotope), and response factor (Rf) of selected compounds used in SIDA are presented in Table 1. The Rf was the reverse value of the

Table 1. Selected Ions (m/z) of Unlabeled and Labeled Compounds and Rf Applied for Quantitation unlabeled

compounds 2-methylpropanal 2-methylbutanal 3-methylbutanal

labeled selected ion (m/z) 72 86 86

compounds

selected ion (m/z)

Rf

[ H2]-3-methylbutanal [2H2]-3-methylbutanal [2H2]-3-methylbutanal

88 88 88

0.21 0.51 1.24

2

slope, which was obtained by plotting the mass ratio (10:1, 4:1, 2:1, 1:1, 1:2.5, and 1:5) and peak area ratio of unlabeled and labeled compounds. Sensory Evaluation. The aroma-liking score of RBPH-F for cocoa-flavored product application was investigated using a 9-point hedonic scale (1 = dislike extremely, 9 = like extremely) with 40 untrained panelists.6 Statistical Analysis. A randomized complete block design was used in all experiments. Data on DH were run in triplicate; data on the amount of Strecker aldehydes (2-MP, 2-MB, and 3-MB) were run in duplicate and were analyzed using analysis of variance (ANOVA) and Duncan’s multiple range tests to separate significant means. Differences were reported as significant at p = 0.05, using SPSS version 12.0 (SPSS Inc., Chicago, USA).

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RESULTS AND DISCUSSION Odor Description of RBPH-F. RBPH-F was chosen to study the odor description by GCO analysis because RBPH-F presented the significantly highest aroma intensity of desirable aroma characteristics from descriptive sensory analysis and overall aroma-liking score.4 The odor descriptions of RBPH-F are demonstrated in Table 2. Thirty-nine odor-active compounds could be detected by GCO. The odor-active compounds were matched with GC-MS results and the C

DOI: 10.1021/acs.jafc.9b02025 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 2. Odor Description of RBPH-F RIa no.

RTX-Wax

DB-5

compounds

description (odor)b

identification methodsc

1 2 3, 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

717 788 925 992 1087 1163 1252 1297 1311 1342 1352 1387 1407 1425 1446 1452 1463 1472 1476 1522 1555 1604 1646 1665 1681 1684 1726 1748 1833 1882 2029 2051 2062 2102 2218 2252 2570 2605