Characterization of Odor-Active Volatiles in Hawthorn Puree Using

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Chemistry and Biology of Aroma and Taste

Characterization of odor-active volatiles in hawthorn puree using thermal desorption system coupled with gas chromatographymass spectrometry-olfactometry and GC-flame photometric detector Jiancai Zhu, and zuobing xiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04636 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018

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Journal of Agricultural and Food Chemistry

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Characterization of odor-active volatiles in hawthorn puree

2

using

3

chromatography-mass

4

GC-flame photometric detector

thermal

desorption

system

coupled

with

spectrometry-olfactometry

gas and

JianCai Zhu1,2, ZuoBing Xiao1*,2

5 6

1

7

Shanghai, China

8

2

Department of Perfume and Aroma Technology, Shanghai Institute of Technology,

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

1

ACS Paragon Plus Environment


9

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Abstract

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In the study, the volatile compounds in hawthorn puree obtained from three

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cultivars (Y1, Crataegus pinnatifida Bunge. cv. ‘Waibahong’, Y2, Crataegus

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pinnatifida Bunge. cv. ‘Damianqiu’, and Y3, Crataegus pinnatifida Bunge. cv.

13

‘Dajinxing’) were analyzed by the thermal desorption system (TDS) coupled with gas

14

chromatography-mass

15

photometric detector (GC-FPD). In the samples of Y1, Y2, and Y3, 40, 42, and 42

16

odor-active compounds were respectively identified by olfactometry. Methyl

17

2-methylbutanoate, methyl hexanoate, hexyl isobutanoate, methional, heptanal and

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(Z)-3-hexenyl acetate contributed largely to the characteristic aroma of the three

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samples. Additionally, the odor activity value (OAV) was used to determine the main

20

compounds

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3-mercapto-2-methylpentanal (OAV: 4-7), methyl 2-methylbutanoate (OAV: 9-21),

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methyl hexanoate (OAV: 8-16), and 2-pentyl acetate (OAV: 5-12) were considered as

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the important contributors to the aroma of hawthorn samples.

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Keywords: Hawthorn puree; TDS; FPD; GC-MS-O; OAV

and

spectrometry-olfactometry

3-mercaptohexyl

(GC-MS-O)

acetate

2


and

(OAV:

GC-flame

10-18),

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Introduction

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Aroma is an important factor to be considered in fruit quality assessment. It is

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mostly influenced by complex volatile compounds, such as alcohols, esters, acids,

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aldehydes, ketones, and terpenoids.1

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Although there are thousands of volatile compounds in fruit, it is well-accepted

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that only a small fraction of volatile compounds occurring in food actually contribute

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to the overall aroma. Thus, the identification of the odor-active compounds was the

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most important task in aroma analysis. GC-O and odor activity value (OAV) were

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considered as effective methods for determining odor-active compounds.

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was widely employed to identify odor-active compounds in fruits, such as blueberry,

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cranberry, mango.2, 4, 5 OAV is the ratio of concentration to the odor threshold of the

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compound. It is well-accepted that compounds with higher OAV contribute more to

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the aroma of the food.

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odorants in foods. 6-9

3

2, 3

GC-O

Thus, OAV has been used widely in determining potent

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Except those compounds with high contents, sulfur compounds with low

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contents might contribute to the characteristic aroma of fruit due to their extremely

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low thresholds. Sulfur compounds widely exist in many foods, such as mulberry,1

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raspberry,10 cheeses,11 ham,12 blueberry,13 grapefruit,14 and cranberry.5 Furthermore,

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sulfur volatile compounds are considered as odor-active compounds. For example,

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Fatima reported the sulfur compounds in grapefruit juice and indicated that

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3-mercaptohexylacetate, 3-mercapto-1-hexanol, 4-mercapto-4-methyl-2-pentanone

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played an important role in the flavor of grapefruit juice.15 Sulfur volatiles in three

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fragrant rice cultivars were investigated by GC-PFPD and 2-acetyl-2-thiazoline,

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dimethyl sulfide, 3-methyl-2-butene-1-thiol, 2-methyl-3-furanthiol, dimethyl trisulfide,

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and methional were confirmed as important odor-active compounds in rice.16 Thus,

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sulfur compounds made important contributions to the aroma of various foods and

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generally determined the characteristic flavors of foods.

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Hawthorn (Crataegus pinnatifida Bunge) has a high nutritional value since it

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contains rich vitamins, flavonoids, organic acids, and microelements.17 It is

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recognized as one of the high-quality raw materials for processing health foods.

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Recently, previous studies on hawthorn mainly focused on volatile compounds,17-19

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polyphenolie profile, biological activity,20-23 and pharmacological properties. 24, 25

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18

However, the systematic evaluation of odor-active volatile compounds in 3



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hawthorn was not reported, especially sulfur compounds. The study aims to determine

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the key aroma compounds in hawthorn by GC-MS-O, GC-FPD and OAV and

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characterize the aroma profile of hawthorn samples by sensory evaluation.

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Experimental

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Chemicals

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All the standard compounds detected by GC-MS and GC-FPD were purchased

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from Sigma-Aldrich (Saint Luis, EUA) and of analytical grade.

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Materials

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Three varieties of hawthorn (Y1, Crataegus pinnatifida Bunge.cv. ‘Waibahong’,

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Y2, Crataegus pinnatifida Bunge. cv. ‘Damianqiu’, and Y3, Crataegus pinnatifida

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Bunge. cv. ‘Dajinxing’) were picked in hawthorn plantation in 2017. About 500 g of

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hawthorn sample was crushed and deseeded. Then, the treated sample was placed in a

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juicer, in which 400 g of deionised water, sodium chloride solution (40g, 20 %) and

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sodium fluoride solution (30 g, 1%) were added to prepare hawthorn puree. Sodium

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chloride was employed to reduce possible enzyme activity, and sodium fluoride was

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used to reduce microbial growth. The puree was kept in a refrigerator at 5 °C.

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Purge and trap of volatile compounds in hawthorn puree

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In this experiment, 6 g of hawthorn puree and 50 μL of the internal standard

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solution containing 10 mg/kg of 1,3-dichlorobenzene (or 10 μL of the internal

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standard solution containing 10 μg/kg of dipropyl disulfide) were prepared in a

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100-mL glass bottle with two ports. One port was coupled with a glass tube with high

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purity nitrogen gas. Another port was connected to Tenax TA extraction tube (Gerstel,

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Mullheim an der Ruhr, Germany). The glass bottle was placed in water bath at 35 °C

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for 40 min. During this extraction process, nitrogen was supplied at a flow rate of 30

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mL/min in order to release volatile compounds in hawthorn puree. Before extracting

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hawthorn puree, the main parameters (extraction time, sample weight, and extraction

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temperature) were investigated. The optimized extraction time, sample weight, and

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extraction temperature were 60 min, 6 g, and 35 °C, respectively. Finally, the

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extraction tube was analyzed with the TDS/CIS-GC-MS-O or TDS/CIS-GC-FPD.

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Conditions of thermal desorption system (TDS)

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TDS-3 was installed on GC and GC-MS equipped with a cooled injector system

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(CIS) (Gerstel, Mullheim an der Ruhr, Germany). Splitless thermal desorption was

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performed. The program temperature was maintained at 40 °C for 1 min, ramped at 4


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the rate of 60 °C/min to 240 °C and maintained for 10 min. The analytes were

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cryo-focused in the CIS at -90 °C with liquid nitrogen and CIS was ramped from -90

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to 240 °C at 12 °C/s.

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GC-MS-O conditions

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GC-MS instrument (7890A-5975C) was employed in the experiment. The

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electron ionization energy of GC-MS was 70 eV. The temperatures of ion source and

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quadrupole were 230 °C and 150 °C, respectively. The scan range was 30-450 m/z.

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Two different columns of DB-Wax and DB-5 (60 m×0.25 mm i.d.×0.25 μm film

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thickness) were used in this experiment. The oven temperature was maintained at 40

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°C (6 min), then ramped to 100 °C (3 °C/min), then ramped to 230 °C (5 °C/min) and

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maintained for 10 min. The split ratio was 5:1. The compounds were determined

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based on NIST 11 Database, standard compounds, and retention indices (RIs). The

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RIs were calculated by alkanes (C6-C30) (Sigma-Aldrich, St. Louis, MO).

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The GC-MS was equipped with an ODP-2 Olfactory Detector Port (Gerstel,

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Mulheim an der Ruhr, Germany) in order to conveniently collect MS signal and odor

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characteristics of each compound effluent from sniffing port. Before the experiment, a

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panel of 10 members (6 males and 4 females) with experience in assessing fruit aroma

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were research laboratory staff at School of Perfume and Aroma Technology, Shanghai

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Institute of Technology. They were trained with standard compounds in hawthorn

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detected by GC-MS in order to master basic aroma descriptions. During the

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experiment, the panelists were asked to quickly note the odor characteristic and aroma

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intensities. A 10-point intensity scale criteria was employed to evaluate the intensity

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of odorants. “0” refers to non-aroma; “5” refers to moderate aroma; “10” refers to

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extreme aroma. They attended 2 sessions per week, each lasting 90 min, for 3 weeks.

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The hawthorn puree were perceived with same process. The experiment was repeated

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three times. To ensure the reliability of the data, the relative standard deviation of

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scoring for three time should be less than 10%. Otherwise, the data were invalid. Last,

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the experimental data were averaged from the data from the panelists.

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TDS-GC-FPD

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The Agilent-7890A GC equipped with TDS and a flame photometric detector

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(FPD) was employed in this experiment. The conditions of TDS, CIS, and oven

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program were similar to those of TDS-GC-MS-O. The temperature of flame

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photometric detector was 250 °C. The photomultiplier voltage was 500 V. The sulfur 5



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compounds were determined with authentic standards and RIs.

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Calibration of standard curves

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In order to establish standard curves, a non-volatile matrix was prepared from

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hawthorn puree. During the experiment, nitrogen (99.99%) was employed to remove

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the volatile compounds in puree. Standard stock solution containing volatile

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compounds detected in hawthorn was also prepared. The dilution was carried out

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according to the ratios of 1:5, 1:10, 1:20, 1:30, 1:40 and 1:50 with Milli-Q deionized

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water. Then, 0.05 g of the diluted solutions containing 50 μL of 10 mg/kg

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1,3-dichlorobenzene as the internal standard of non-sulfur compounds was added into

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6 g of non-volatile matrix. Similarly, 0.05 g of the diluted solutions containing 10 μL

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of 10 mg/kg dipropyl disulfide as the internal standard of sulfur compounds were

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added into 6 g non-volatile matrix. The extraction conditions were similar to the

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conditions of thermal desorption system (TDS). After that, the standard curves were

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established. The concentrations of volatile compounds in puree were determined

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according to standard curves, in which “y” was the ratio of the peak area of volatile

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standard to the peak area of internal standard and “x” was the ratio of the

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concentration of volatile standard to the concentration of internal standard.

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Odour activity values (OAV)

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The contribution of compound to the hawthorn aroma was determined by the

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odor activity value (OAV), which was defined as the ratio of the concentration of each

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compound to its detection threshold in water. 26 The threshold values were taken from

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information available in the references. 1, 27, 28

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Quantitative descriptive sensory analysis

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A panel of 10 members (5 males and 5 females) were employed to evaluate the

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sensory profile of hawthorn puree. All panelists were research laboratory staff at

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School of Perfume and Aroma Technology, Shanghai Institute of Technology. They

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attended 2 sessions per week, each lasting 90 min, for 3 weeks. Firstly, hawthorn

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puree was added in a 20-mL vial. Then, the vial was assigned to the panelists at

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30 °C. After that, the organoleptic characteristic of hawthorn puree was determined as

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six attributes: “floral”, “sour”, “green”, “fat”, “fruity” and “sulfur”. The scores were

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obtained according to 10-point scales. “0” refers to non-aroma; “5” refers to moderate

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aroma; “10” refers to extreme aroma. The aroma of “floral” descriptor refers to

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β-ionone; “sour” descriptor refers to acetic acid; “green” descriptor refers to

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(E)-2-hexenal; “fat” descriptor refers to nonanal; “fruity” descriptor refers to ethyl 6


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hexanoate; “sulfur” descriptor refers to dipropyl disulfide. The experiment was

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repeated three times.

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Intensity ratings were made using a modified of previous literature.29 In the

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sensory evaluation experiment, butanol was selected as the reference. In order to

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cover the intensity range of all used odors, five butanol solutions of different

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concentrations were prepared in the experiment. The sensory panelists were

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continuously sniffed and remembered the intensity values of different concentrations

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of butanol solution. Before each sensory evaluation, the panelists sniffed the intensity

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of butanol at different concentrations. In order to better match the perceived strength

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with the strength of 1-butanol, the intensity scale of 0-10 (zero intensity to strong

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intensity) was adopted in the experiment. The intensities of 1-butanol of 5 positions

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were respectively corresponded to 5 levels. The mean value of each sample was

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presented by the triplicate means score based on ten point scales.

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Impact of compounds added into aromatic reconstitution (AR)

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Firstly, three-alternative forced-choice presentation (3-AFC) was employed to

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measure the olfactory thresholds of aromatic reconstitution. The AR consisted of

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volatile compounds (methyl 2-methylbutanoate, 3-mercaptohexyl acetate, acetic acid

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and undecanal) detected in sample Y1 at the actual concentrations. Then, AR was

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evaluated by well-trained panelists containing 8 males and 7 females in 3-AFC tests,

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respectively. The volume represented the dilution degree of AR solution, which were

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diluted from 0.1 mL to 51.2 mL at a factor of 2. The volume/response function was a

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psychometric function and the detection threshold was determined by a sigmoid

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curve. In the previous findings, the detection threshold was defined as the

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corresponding concentration (volume), at which the correct detection probability was

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50%. Finally, the compounds methyl 2-methylbutanoate, 3-mercaptohexyl acetate,

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acetic acid and undecanal with actual concentrations detected in Y1 sample were

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added into AR, respectively. The olfactory thresholds of those four mixtures were

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evaluated according to the same procedure. The more detailed procedure can be found

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in previous studies.30

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Statistical analysis

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The data of aroma intensity and concentration of volatile compounds were

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processed with the analysis of variance (ANOVA) with XLSTAT 7.5 (Addinsoft,

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New York, NY, USA). Duncan’s multiple range tests were performed to determine

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significant differences (p

Characterization of Odor-Active Volatiles in Hawthorn Puree Using

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