Study of the Inhibitors of Cooked Off-Flavor Components in Heat

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Chemistry and Biology of Aroma and Taste

Study of the Inhibitors of Cooked Off-Flavor Components in Heat-Treated XiZhou Melon Juice DongSheng Luo, Xinxing Xu, Shuang Bi, Yuping Liu, and JiHong Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03398 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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Journal of Agricultural and Food Chemistry

Study of the Inhibitors of Cooked Off-Flavor Components in Heat-Treated XiZhou Melon Juice Dongsheng Luo1, Xinxing Xu1, Shuang Bi1, Yuping Liu2, Jihong Wu1 1. College of Food Science and Nutritional Engineering, China Agricultural University; Key Laboratory of Fruit and Vegetable Processing, Ministry of Agriculture; National Engineering Research Center for Fruit and Vegetable Processing, Beijing 100083, China 2. Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business University, Beijing 100048, China

1

Corresponding author. Tel/fax: +86-010-62737434-603. E-mail: [email protected]

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Abstract: This research applied inhibitors to reduce the content of cooked off-flavor

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components (dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, and

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3-(methylthio)propanaldehyde) in heat-treated melon juice. The effects of glucose

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oxidase (GOD) on the formation and release of these four volatile sulfur compounds

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were also investigated. Results showed that GOD strongly inhibited the formation of

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the four compounds. In GOD-treated melon juice, S-methylmethionine was strongly

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protonated and not easily degraded into dimethyl sulfide. Moreover, the release of the

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dimethyl sulfide that did form was restrained by the hydrophobic interactions of

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gluconic acid and oxidation by hydrogen peroxide. In addition, gluconic acid (or

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glucose) and hydrogen peroxide could form a stable complex with methionine in an

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acidic

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3-(methylthio)propanaldehyde, dimethyl disulfide, and dimethyl trisulfide by the

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Maillard reaction during heat processing.

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Key words: melon juice; cooked off flavor; inhibitor; glucose oxidase; mechanisms

matrix

and

thus

prevented

the

methionine

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from

producing

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Introduction

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Melon juice, a common deep-processed product, is convenient to transport and store,

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helping to avoid postharvest loss of melon fruits.1 Volatile sulfur compounds (Figure

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1), such as dimethyl sulfide (1), dimethyl disulfide (2), dimethyl trisulfide (3), and

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3-(methylthio)propionaldehyde (4), are major contributors to off flavors in

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heat-treated melon juice.2-4 The production of these compounds seriously degrades the

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flavor quality of melon juice. However, some heat treatments, like heat sterilization,

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which can kill and inactivate microorganisms and enzymes in juice, are very

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important for the safety and storage of melon juice. Therefore, controlling the

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development of the volatile sulfur compounds in heat-treated melon juice is necessary

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for the deep processing of melon fruits.

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S-methylmethionine (SMM) and methionine (Met) are the two main flavor precursors

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of the four volatile sulfur compounds that form in melon juice.4 However, these

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precursors are almost impossible to eliminate completely by common separation

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techniques (such as ultrafiltration), while guaranteeing the good quality of the melon

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juice. Moreover, they are micronutrient elements in juices and can't be removed easily.

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Thus, use of inhibitors is a practical method to improve the flavor quality of the

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heat-treated melon juice. Lermusieau, (2015) reported that the content of compound 1

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content in boiling wort could be reduced with an acid additives.5 Phenol compounds,

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especially, polyphenols can trap reactive dicarbonyls to control the development of

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off-flavor components through the Maillard reaction in ultrahigh heat-treated milk.6

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Cysteine, pyridoxine, and thiamine can scavenge free radicals (or reactive dicarbonyls)

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and compete to react with the amino acids commonly used as Maillard reaction

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inhibitors in the food industry.7 In addition, phenolic acid, ascorbic acid, and glucose

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oxidase (GOD) can restrain the Maillard reaction and heat degradation of SMM by

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reducing free radicals (or reactive dicarbonyls), and decreasing the dissolved oxygen,

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glucose content, and the pH values of the food matrix.8-10 Nevertheless, few reports on

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improving the flavor quality of juice using these inhibitors are currently available. The

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functional mechanisms of some inhibitors on the formation of compounds 1, 2, 3, and

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4 in juices during heat processing are unknown.

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Thus, this study aimed to (1) screen for the optimal inhibitor that can simultaneously

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control the heat degradation of SMM and Met in melon juice; (2) analyze the effect of

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the main changed components in melon juices on the release of compound 1; and (3)

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explore the molecular mechanisms underlying the inhibition of the formation of

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compounds 1, 2, 3, and 4 during heat processing.

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Materials and Methods

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Chemicals

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N-alkanes (C5-C30), L-methionine (Met, CAS: 59-51-8), S-methylmethionine (SMM,

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CAS: 4727-40-6), dimethyl sulfide (1, CAS: 75-18-3), dimethyl disulfide (2, CAS:

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624-92-0), 3-(methylthio)propanaldehyde (4, CAS: 3268-49-3), dimethyl trisulfide (3,

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CAS: 3658-80-8), L-homoserine (HS, CAS: 672-15-1), homoserine lactone

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hydrochloride (HSL, CAS: 2185-03-7), dimethyl sulfoxide (DMSO, CAS: 67-68-5),

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gluconic acid (CAS: 526-95-4), and glucose (CAS: 50-99-7) were purchased from

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Sigma–Aldrich Co., Ltd. (Milwaukee, WI, USA) with purity>98%. Ammonium

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hydroxide solution (purity>25%), formic acid (purity>99%), and acetonitrile

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(purity>99%) were obtained from Merck & Co., Inc., (Kenilworth, NJ, USA).

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Glucose oxidase (GOD, 250 U/mg), hydrogen peroxide (w/w, 35%), sodium

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dihydrogen phosphate, and disodium hydrogen phosphate were purchased from

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Beijing Solarbio Science & Technology Co., Ltd (Beijing, China).

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Melon samples

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XiZhou melon (C. melo var. Reticulates, 50 kg) was purchased directly from Xinjiang

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Uigur Autonomous Region of China in June 2018. Reducing sugar content was about

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7.0% (w/w) and pH value was at 5.2–5.6.

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Sampling melon juice and model solutions

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Melon fruits were maintained in an ice bath for 12 h (to reduce the deterioration of

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their quality during processing) and then squeezed. The juice was centrifuged at

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11000 ×g for 10 min at 4 °C. Clear melon juice was prepared from the supernatant by

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using an ultrafiltration unit (pore diameter 50 nm).4

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The blank solution was an aqueous solution with a pH of 5.2 (adjusted by phosphates).

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The glucose and gluconic acid solutions were 7 g glucose/100g blank solution and

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2.33 g gluconic acid/100g blank solution, respectively. The mixture of glucose and

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gluconic acid solution was 4.67 g glucose and 2.33 g gluconic acid/100g blank

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solution. The model solutions of compound 1 were 9.0 mg 1/L blank solution, glucose

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solution, gluconic acid solution, or the mixture solution. The SMM and Met model

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solutions were 7 mg SMM/L glucose solution and 70 mg Met/L glucose solution,

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respectively.

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Inhibitor and heat treatment

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Clear melon juice (10 mL) and inhibitors (5 mg/5 U, ferulic acid, chlorogenic acid,

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rosmarinic acid, protocatechuic acid, gallic acid, epicatechin, epicatechin gallate, rutin,

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quercetin, resveratrol, cysteine, thiamine, ascorbic acid, pyridoxamine, and GOD)

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were added to a flask. The flask was sealed and shaken at 200 rpm for 160 min (30

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°C).

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a UHT/HTST processing system in accordance with the method reported by Luo et al.

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(2018).4 Met or SMM model solutions were treated by GOD and heat treatment

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following the method previously described.

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Determination of inhibition ratios

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Sample (6 mL) and sodium chloride (3 g) were transferred into screw-cap headspace

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vials (22.8 mL, Chromoptic, France). The detection of compounds 1, 2, 3, and 4 in

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samples through headspace solid-phase microextraction-gas chromatography-mass

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spectrometry (HS-SPME-GC-MS) was performed as described by Luo et al. (2018).4

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A 50/30 m polydimethylsiloxane/divinylbenzene/carboxen coated SPME fiber (2 cm)

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was exposed to vial headspace for 30 min at 40 °C with agitation at 100 rpm after 10

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min of equilibration. Then, the SPME fiber was inserted into a GC-MS injection port

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(7890B/5975C, Agilent Technologies, Inc., Santa Clara, CA, USA) at 250 °C for 5

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min. Volatile components were separated on DB-5MS capillary columns (30 m × 0.25

Then, the samples were subjected to heat treatment (130 °C, holding for 3 s) using

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mm i.d. × 0.25 m; J&W Scientific, Folsom, CA, USA).

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The temperature program was initially held for 2 min at 35 °C, then increased to 150

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°C

at the rate of 4 °C /min, increased to 250 °C at the rate of 10 °C /min, and held for 5

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min. MS was performed in electronic impact mode (70 eV). The ion source

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temperature was 250 °C with selected ion monitoring mode (SIM).

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The compounds 1, 2, 3, and 4 were positively identified in reference to the National

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Institute of Standards and Technology mass spectrometer library (match quality

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>80%), retention index (504, 736, 906, and 968), and standard substances.

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The change of contents of compounds 1, 2, 3, or 4 in samples was evaluated using the

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precise peak areas of each compound’s characteristic ion acquired by SIM, given the

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identical detector response factor of each sulfur compound. The characteristic ions of

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compounds 1, 2, 3, and 4 are 62, 94, 126, and 104, respectively.4

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The inhibition ratios for compounds 1, 2, 3, and 4 in heat-treated melon juice were

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calculated as follows:

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Inhibition ratio (%) 1

Ats 100% As

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where Ats and As are the peak areas of the characteristic ions of 1, 2, 3, or 4 in treated

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and untreated melon juice, respectively, after heat treatment.

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Analysis of SMM/Met and degradation products

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The SMM or Met model solutions (1 mL) after treatment were purified using a

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Cleanert-PEP SPE column (60 mg/3 mL, Phenomenex and Agela Technologies, CA,

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USA) after centrifugation (11000 ×g, 10 min).11 The eluent from SPE column was

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filtered with disposable sample filters (0.22 m) and collected for analysis with an

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ultra performance liquid chromatography system coupled to a triple quadrupole mass

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spectrometer (UPLC-MS/MS) (Waters ACQUITYI-CLASS, Waters Co., Milford,

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MA, USA). Separation was performed using the Acquity UPLC BEH C18 column

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and Acquity UPLC BEH Amide column (100 mm × 2.1 mm, 1.7 m particle size).

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The solvent system for the C18 column consisted of 0.1% aqueous formic acid (A)

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and acetonitrile (B) with gradient elution at a flow rate of 0.4 mL/min. The system

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was used as follows: 0– /R2

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3.0–1 R2

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was used in positive mode through full scanning mode and multiple reaction

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monitoring mode. The ion source capillary voltage and cone voltage were 3.5 kV and

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35 V, respectively. The optimized selected MS/MS transition pairs of the precursor

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and product ions were as follows: Met 150>133 and 150>104 (collision voltage 9 and

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10 V), SMM 164>102 (collision voltage 12 V), homoserine 120>74 (HL, collision

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voltage 11 V), and homoserine lactone 102>74 (HSL, collision voltage 10 V).

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The solvent system for the amide column consisted of 0.1% aqueous ammonia (A)

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and acetonitrile (B) with gradient elution at a flow rate of 0.2 mL/min and was used as

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follows: 0– /R2

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5.1–0 /R2

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by full scanning mode (50–1000 m/z) and daughter mode (collision voltage 12 V).

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The other MS parameters were the same as those described by Luo et al. (2018).4

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Compounds were identified and quantified through comparison with the retention

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times, MS spectra, and MS/MS fragmentation patterns of standard substances. The

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contents of SMM, HS, and HSL in the model solutions were expressed in terms of the

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peak areas of their ion pairs.

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Measurement of hydrogen peroxide (H2O2) in GOD-treated melon juice

1% B; 1.0– BR2

95%–1% B; and 3.1–0 /R2

1%–95% B; 1.5–1 /R2

95% B;

1% B. The electron spray ionization source

90%–60% B; 2.0–B /R2

60% B; 5.0–B R2

60%–90% B; and

90% B. The electron spray ionization source was used in negative mode

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H2O2 in GOD-treated melon juice was detected according to the method described by

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Zhao et al., (2019) with minor modification.12 GOD-treated melon juice (processing

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time at 20, 40, 60 80, 100, 120, 140, or 160 min, 6 g) was blended with precooled

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acetone (6 mL). The mixture was shaken for 3 min and then centrifuged at 11000 ×g

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at 4 °C for 5 min. Then, the supernatant (1 mL) was mixed with 20 mmol/L titanium

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sulfate (0.1 mL) and ammonia (0.2 mL). The mixture was centrifuged at 11000 ×g at

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4 °C for 5 min. After removal of the supernatant, the residue was dissolved in 1 mol/L

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sulfuric acid (3 mL). The mixture was shaken for 3 min and then centrifuged at 11000

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×g at 4 °C for 5 min. The absorbance of the supernatant was measured at 415 nm. For

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the calibration curve, a series of H2O2 standard solutions was prepared in ultrapure

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water at concentrations of 2.03, 4.11, 8.23, 16.15, 32.06, and 64.27 mmol/L

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(y=0.61x+0.052, r2=0.9859).

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Detection of dimethyl sulfoxide (DMSO) in model solution

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H2O2 solution (100 L) and compound 1 glucose model solution (100 mL) were

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transferred into a 150 mL screw cap glass vial. The sealed vial was shaken at 200 rpm

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for 128 min. Then, the mixture (processing time at 1, 2, 4, 8, 16, 32, 64, or 128 min)

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was centrifuged at 11000 ×g for 5 min. The supernatant was filtered with disposable

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sample filters (0.22

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chromatography equipped with a diode array detector (DAD) (HPLC, Agilent 1260,

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Agilent Technologies, Inc., Santa Clara, CA, USA).13 Samples were separated with

167

the Venusil MP C18 column (4.6 mm × 250 mm). The solvent system consisted of

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aqueous and acetonitrile (6:94). The flow rate was 1.0 mL/min, and column

m). DMSO was analyzed by high-performance liquid

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temperature was 35 °C. For the calibration curve, a series of standard solutions of

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DMSO were prepared in ultrapure water at concentrations of 3.125, 12.5, 50, 200, and

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800 mg/L (y=0.26x-0.038, r2=0.9937).

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Determination of partition coefficients of dimethyl sulfide (1)

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Different amounts of compound 1 model solution (0.5, 1, 2, 3, 4, or 5 mL) were

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transferred into a 22.8 mL screw cap headspace vial with phase ratios of 45.6 to 4.56

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(according to the model solution volumes). The samples were analyzed by

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HS-GC/MS.14 The headspace vial was equilibrated for 120 min at 35 °C. After

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shaking, a 500 L sample of the headspace was withdrawn with a 2.5 mL thermostatic

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gastight syringe, and preheated to 45 °C on a Gerstel autosampling device (Mülheim

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an der Ruhr, Germany). The syringe was inserted into the GC-MS injection port at a

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rate 0.5 mL/s. The injection port was held at 250 °C with a split ratio of 1:3. The

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conditions of the GC and MS were the same as those described above

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(HS-SPME-GC/MS). Partition coefficients of compound 1 in different model

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solutions were calculated via the method developed by Ettre et al. (1993)15: 1 A

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1 liq i

fi C

1 fi Ciliq

kg/m

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By plotting 1/A against , this equation gives a linear relationship between 1/A and ,

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as follows:

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1 A

a

b*

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where kg/m is the partition coefficient between the gas and the matrix (namely, b/a), A

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is the chromatographic peak area of compound 1, fi is the detector response factor,

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Ciliq is the initial concentration of the compound in the vial, and

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the headspace (Vg) and matrix (Vl) volumes.

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Analysis of interaction between compound 1 and glucose or gluconic acid

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The changes in energy during the interaction of compound 1 and glucose or gluconic

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acid were determined with an isothermal titration calorimeter (ITC-200 MicroCal, GE,

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Northampton, USA).16 Compound 1, glucose, and gluconic acid solutions (11.21, 0.56,

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and 0.56 mmol/L, respectively) were prepared in 1% propylene glycol aqueous

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solution (v/v) at pH 5.2 (adjusted by phosphates). The solutions were degassed by

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ultrasound for 5 min and filtered with disposable sample filters (0.22 m). The sample

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cell was filled with 300 L glucose or gluconic acid solution and titrated with 50

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of compound 1 model solution placed in the stirring syringe. Experiments were set up

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with 24 consecutive injections (2.02 L) with a duration of 10 s each, at intervals of

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150 s, a stirring speed of 300 rpm, and temperature fixed at 25 °C.

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Statistical analysis

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All experiments were conducted in triplicate. Data analysis was performed using

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SPSS software (v17.0, Chicago, IL, USA). When differences between treatments

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(peak area of 1, 2, 3, 4, HS, HSL and SMM with a 95% confidence interval) were

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statistically significant, means were compared through Duncan’s multiple range test at

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the significance level of p0.05) but SMM content obviously changed (p