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Effect of processing on the phenolic contents, antioxidant activity and volatile compounds of sorghum grain tea
Accepted Manuscript Effect of processing on the phenolic contents, antioxidant activity and volatile compounds of sorghum grain tea Yun Xiong, Pangzhen Zhang, Jiaqian Luo, Stuart Johnson, Zhongxiang Fang PII:
S0733-5210(18)30614-3
DOI:
https://doi.org/10.1016/j.jcs.2018.10.012
Reference:
YJCRS 2659
To appear in:
Journal of Cereal Science
Received Date: 9 August 2018 Revised Date:
19 September 2018
Accepted Date: 31 October 2018
Please cite this article as: Xiong, Y., Zhang, P., Luo, J., Johnson, S., Fang, Z., Effect of processing on the phenolic contents, antioxidant activity and volatile compounds of sorghum grain tea, Journal of Cereal Science (2018), doi: https://doi.org/10.1016/j.jcs.2018.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Effect of processing on the phenolic contents, antioxidant activity and volatile
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compounds of sorghum grain tea
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Yun Xiong1, Pangzhen Zhang1, Jiaqian Luo1, Stuart Johnson2, Zhongxiang Fang1*
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Melbourne, Parkville, VIC 3010, Australia
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University, Perth, WA 6845, Australia
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School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, University of
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School of Molecular and Life Sciences, Curtin Health Innovation Research Institute, Curtin
*Corresponding author: Dr Zhongxiang Fang
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School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, University of
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Melbourne, Parkville, VIC 3010, Australia
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Email: [email protected]; Tel: +61 3 83445063
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Abstract: Sorghum grain is rich in phenolic compounds and may be used to develop functional tea
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beverages. This work investigated the effect of processing techniques on the phenolic contents,
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antioxidant activity, and volatile compounds of a white colour sorghum (Liberty) grain tea.
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Significant (P ≤ 0.05) increase of total phenolic content, total flavonoid content and condensed
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tannin content were observed during the processing, whereas the antioxidant activity was not
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statistically enhanced. A total of 63 volatile compounds were detected including 5 alcohols, 13
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alkanes, 2 aldehydes, 2 carboxylic acids, 15 esters, 4 ketones, 3 pyrazines and 1 phenylenediamine,
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which were affected by the processing techniques. The sorghum tea made from powder form
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infusion had more abundant volatile compounds compared to whole grain form infusion. The
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findings of this research have potential to expand human consumption of sorghum grain in the new
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form of grain tea.
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Keywords: Sorghum grain tea; processing technology; phenolic content; volatile compounds.
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1. Introduction
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Sorghum (Sorghum bicolor L. Moench) is the fifth leading cereal crop in the world (Wu et al.,
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2013). It contains high levels of bioactive phytochemicals, particularly phenolic compounds such as
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phenolic acids, flavonoids and condensed tannins (Awika & Rooney, 2004; de Morais Cardoso et
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al., 2017). Studies have shown that sorghum grain has high antioxidant activity, good cholesterol-
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lowering ability, anti-inflammatory and anti-carcinogenic properties (de Morais Cardoso et al.,
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2017; Dykes & Rooney, 2006; Stefoska-Needham et al., 2015). It is suggested that regular
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consumption of sorghum grain have the potential of reducing the risk of cancers, cardiovascular
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diseases and type II diabetes (Awika & Rooney, 2004; Okarter & Liu, 2010).Therefore, there is an
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increased interest using sorghum grain to develop functional foods. Recently, application of cereal
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grain to make grain tea beverage is a new trend. For example, Tartary buckwheat, barley and brown
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rice tea have been developed as functional tea beverage owing to their unique flavour, taste and
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health-promoting benefits (Guo et al., 2017; Yuki, 2017). Sorghum grain could be an idea material
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to be developed into grain tea. The additional advantage of using sorghum grain as tea making
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material is that sorghum is a sustainable grain that could be grown in a semi-arid environment
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(Stefoska-Needham et al., 2015) and the cost of sorghum grain is much lower compared to leaf tea.
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However, limited research has been conducted on sorghum grain tea processing with only one paper
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investigating the phenolic compositions and biological activities of a red sorghum grain tea (Wu, et
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al., 2013).
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Consumers’ satisfaction of tea is affected by its quality, in which aroma is a key quality indicator
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(Kraujalytė, Pelvan, & Alasalvar, 2016). Therefore, it is critical for the food industry to understand
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the aroma profile of sorghum grain tea and how it is affected by the processing technology. The
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aroma profile of raw sorghum grain has been recently reported by Zanan, Khandagale, Hinge,
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Elangovan, Henry, and Nadaf (2016), where a total of 29 volatile compounds were detected in two
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sorghum varieties of E228 and M35-1. In another research, Chughtai, Pasha, Anjum, and Nasir 2
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(2015) detected a total of 35 volatile compounds from seven commercial sorghum varieties.
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However, no information is currently available about the volatile profile of sorghum grain tea.
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Therefore, the objective of this study was to investigate the effect of sorghum grain tea processing
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techniques (soaking, steaming and roasting) on the phenolic contents, antioxidant activity and
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aroma profile. To our knowledge, this research is the first to report volatile profiles of sorghum
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grain tea product and the influences of processing techniques on volatile composition.
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58 2. Materials and Methods
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2.1. Processing of sorghum grain tea
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A non-tannin white colour sorghum variety (Liberty) was supplied by Nuseed (Queensland,
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Australia). The selection of this variety is because it is the main commercial sorghum for human
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consumption in Australia. The sorghum grain tea was produced through three consecutive processes:
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soaking, steaming and roasting, as described by Wu et al. (2013) with some modifications (Fig. 1).
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The steaming and roasting processes were both carried out on a commercial oven (Convotherm 4
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easy Dial 10.10 multifunction oven, Wolfratshausen, Germany). During the process, raw, soaked,
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steamed and roasted sorghum grains were sampled and stored in the dark at –20 °C until analysis.
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2.1.1. Soaking
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About 200 g raw whole sorghum grain was used in each replicate and 3 replicates were conducted.
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The raw sorghum was soaked in water (1:4 w/v) for 17 h at room temperature. The soaked sorghum
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grain was washed, drained, and 50 g of the soaked sample were collected for analysis, with the
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remaining sent for steaming process.
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2.1.2. Steaming
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About 100 g of the soaked sorghum grain was steamed at 100 °C for 50 min, then about 50 g of the
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steamed sorghum grain was cooled and collected for analysis, and the remaining was sent for
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roasting process.
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80 2.1.3. Roasting
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About 50 g of the steamed sorghum grain was roasted at 150 °C for 60 min, cooled, collected and
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subjected to analysis.
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84 2.2. Chemicals and materials
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Folin & Ciocalteu phenol reagent, gallic acid, aluminium chloride, (+)-catechin hydrate, vanillin,
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2,2-Diphenyl-1-picrylhydrazyl (DPPH), (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
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acid (Trolox), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),
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SPME fibre assembly polydimethylsiloxane/divinylbenzene (df 65 µm, Fused Silica/SS)
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(PDMS/DVB), potassium persulphate, n-alkanes (C8–C22), 4-octanol, 500 µm fine test sieve, and
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9.00 cm Whatman filter paper (No. 4) were all purchased from Sigma-Aldrich (Castle Hill, NSW,
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Australia). Methanol, sodium carbonate anhydrous, sodium nitrate, and sodium hydroxide pellets
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were from Chem-Supply (Gillman, SA, Australia). Hydrochloric acid 32% was obtained from
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Science Supply (Mitcham, VIC, Australia). Headspace screwtop clear vials (20 ml) and magnetic
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PTFE/sil hdsp cap were purchased from Agilent Technologies (Singapore). All chemicals were of
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analytical reagent or HPLC grade.
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2.3. Analysis of phenolic contents and antioxidant activity
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2.3.1. Sample preparation About 20 g of each collected sorghum grain sample was freeze-dried at -20°C for 6 days. Then 5 g
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of each dried sorghum grain sample was separately ground and sieved (100% through a 500 µm
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sieve) to obtain sorghum powder. All the results in this study were reported on a dry matter basis.
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103 2.3.2. Extraction of phenolic contents
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The extraction of phenolic compounds was performed using the method described by Wu et al.
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(2013) with slight modifications. Briefly, dried sorghum powder (1 g) was extracted with 45 ml of
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80% methanol solution (v/v) for 1 h at 50 °C, and then centrifuged at 3,220 g and 4°C for 10 mins.
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After centrifugation, the supernatant was collected, and the residue was re-extracted one more time
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under the same conditions. The supernatants were pooled and evaporated at 50 °C to dryness using
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a rotary evaporator, then reconstituted in 10 ml methanol to give the phenolic extract solution. The
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extract solution was then stored at -20 °C in dark until analysis.
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2.3.3. Determination of total phenolic content (TPC)
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TPC was measured using the Folin-Ciocalteu method described by de la Rosa, Alvarez-Parrilla, and
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Shahidi (2010). An aliquot of 60 µl of the extract solution and 60 µl methanol were mixed with 750
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µl of 10% Folin-Ciocalteu’s reagent solution (v/v) and incubated for 2 mins at room temperature.
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Then, 600 µl of 7.5% (w/v) sodium carbonate solution was added, and the mixture was incubated at
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50 °C for 20 min in the dark and then cooled to room temperature. The absorbance was measured
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by a UV-Vis spectrophotometer (M501 Single Beam Scanning, Camspec, Leeds, UK) at 760 nm.
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Gallic acid was used as the standard and methanol as blank, and the results were expressed as mg
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gallic acid equivalents (GAE)/g dry basis (db).
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2.3.4. Determination of total flavonoid content (TFC)
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TFC was measured by the aluminium chloride colourimetric method according to de la Rosa,
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Alvarez-Parrilla, and Shahidi (2010) with a slight modification. An aliquot of 150 µl of the extract
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solution was mixed with 660 µl of water and 45 µl of 5% NaNO2 solution (w/v) and incubated for 5
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min at room temperature. Then 45 µl of 10% AlCl3 solution (w/v) was added to the mixture and
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incubated for 3 min at room temperature. Next, 600 µl of 0.5 M NaOH solution was added, and the
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mixture was incubated for 30 min at room temperature in the dark. The absorbance was measured
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by the M501 UV-Vis spectrophotometer at 415 nm. (+)-catechin hydrate was used as the standard
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and methanol as the blank, and the results were expressed as mg catechin equivalents (CAE)/g db.
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CTC was determined by the vanillin-HCl assay method described by Wu et al. (2016) with a slight
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modification. The vanillin-HCl reagent was prepared daily, by mixing an equal volume of 2%
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vanillin in methanol solution (w/v) with acidified methanol solution (8% HCl, v/v). An aliquot of
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800 µl of the extract was mixed with 0.700 µl of vanillin-HCl reagent. The resulting solution was
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mixed and incubated for 20 min at room temperature, and the absorbance was measured at 500 nm
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by the M501 UV-Vis spectrophotometer. Catechin was used as the standard and methanol as the
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blank, and the results were expressed as mg catechin equivalents (CAE)/g db.
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2.3.6. Determination of DPPH radical scavenging activity (DPPH)
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Radical DPPH scavenging activity was measured according to the method of Wu, Johnson,
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Bornman, Bennett, and Fang (2017) with a slight modification. About 24 mg of DPPH was added
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to 100 ml methanol to prepare the stock solution, and then stored at -20°C in the dark. About 8 ml
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stock solution was diluted with 40 ml methanol to prepare the working DPPH solution with an
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absorbance of 1.1 ± 0.02 units at 515 nm. The sorghum extract was diluted ten times with methanol,
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then 0.150 ml of the diluted sorghum extract was mixed with 1.350 ml freshly prepared the DPPH 6
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working solution. The resulting solution was incubated for 8 h at room temperature in the dark, and
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the absorbance was measured at 515 nm. Trolox was used as the standard and methanol as the blank,
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and results were expressed as mg Trolox equivalents (TE)/g db.
152 2.3.7. Determination of ABTS radical scavenging activity (ABTS)
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The ABTS assay was conducted following the method of Wu et al. (2017). Equal amounts of 7.4
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mM ABTS•+ solution and 2.6 mM potassium persulphate solution were mixed and reacted for 12 h
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at room temperature in the dark to give the stock solution. The ABTS•+ working solution was
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freshly prepared, by diluting 0.8 ml of the stock solution with 50 ml methanol to get an absorbance
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of 1.1 ± 0.02 units at 734 nm, measured by the M501 UV-Vis spectrophotometer. The sorghum
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extract was diluted 10 times with methanol, then 0.150 ml of the diluted sorghum extract was
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reacted with 1.350 ml ABTS•+ working solution for 2 h at room temperature in the dark, and then
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the absorbance was measured at 734 nm. Trolox was used as the standard and methanol as the blank,
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and results were expressed as mg Trolox equivalents (TE)/g db.
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2.4. Analysis of volatile compounds
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2.4.1. Moisture content
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Moisture content was measured by drying 3 g of each sorghum sample in an oven at 105 °C until a
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constant weight was reached. Moisture content was used to quantify the volatile compounds on the
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dry basis.
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2.4.2. Sample preparation and headspace solid-phase microextraction (HS-SPME) procedures
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The extraction of volatile compounds was carried out by HS-SPME method, using a 65 µm
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PDMS/DVB fibre equipped with an SPME PAL 3 multi-purpose automated sampler (Agilent,
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USA). Before analysis, the fibre was pre-conditioned at 250 °C for 30 min in the GC injection port
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in accordance with manufacturer’s specifications. 7
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The sorghum volatile compounds were extracted according to the method of Zanan et al. (2016).
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Raw, soaked, steamed and roasted sorghum grains were ground into powder with liquid nitrogen.
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Two gram of sorghum powder was weighted and immediately transferred into a 20-ml headspace
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vial, added with 20 µL of internal standard 4-octanol (0.01 g/100 ml) and then tightly sealed with a
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magnetic PTFE/sil cap. The vial was shaken incubated at 80 °C for 20 min for equilibrium, and the
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SPME fibre was then exposed to the headspace at 80 °C for 35 min for adsorption.
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To analyse sorghum tea volatile compounds, both roasted sorghum grain and its ground powder
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were infused by boiling water as described by Lv, Zhong, Lin, Wang, Tan, and Guo (2012). Briefly,
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about 2 g of whole roasted grain or ground powder was weighted and immediately transferred into a
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20-ml headspace vial, added with 20 µL of internal standard 4-octanol (0.01 g/100 ml) and infused
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with boiling water (10 ml, 100 °C) respectively. The vial was tightly sealed immediately with a
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magnetic PTFE/silicon cap, agitated for 10 min to reach equilibrium. Then, the SPME fibre was
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exposed to the headspace and kept at 60 °C for 60 min.
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2.4.3. Gas chromatography-mass spectrometry (GC-MS) analysis
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The GC-MS analysis was carried out following the method described by Lv, Zhong, Lin, Wang,
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Tan, and Guo (2012) with slight modifications. A gas chromatograph (Agilent Technologies 6850
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series II gas chromatograph, USA) was coupled to a mass spectrometer (Agilent Technologies 5973
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mass selective detector, USA) and a J&W DB-5ms capillary column (30 m x 250µm x 0.25µm)
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was used.
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After extraction, the SPME fibre was introduced to the GC-MS analysis. SPME fibre desorption
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was conducted at GC injection port in splitless mode, with an injection temperature 250 °C. Helium
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(percentage purity > 99.999%) was used as the carrier gas at a constant flow velocity of 1 ml/min. 8
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The oven temperature program was set as follows: an initial temperature of 50 °C and held for 5
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min, then increased to 125 °C at a rate of 3 °C/ min and held for 3 min; and then increased to
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180 °C at a rate of 2 °C/min and held for 3 min; and last increased to the final temperature 230 °C at
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a rate of 15 °C/min and held for 3 min. The mass spectrometer was operated in ionisation mode at
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70 eV, with a mass scan range of 35-400 AMU; the interface, ion source and quadrupole
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temperature were 280 °C, 230 °C and 150 °C, respectively. The solvent delay time was 3.0 min.
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2.4.4. Compound identification and quantification
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The GC-MS data were analysed using Agilent G1701EA MSD ChemStation software (Version
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1.4.20.0). The linear retention index (RI) of each compound was calculated using a series of n-
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alkanes (C8–C22). Each compound was identified by comparing the mass spectra and linear
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retention indices (RI), using the NIST reference database (NIST 11.0) and NIST Chemistry
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Webbook (NIST, 2017), where available. The concentration of each compound was reported semi-
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quantitatively as internal standard (I.S.) equivalents to compare relative concentration changes
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among the studied samples only. The spectrums were scanned in the total ion chromatogram (TIC)
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mode. Each identified volatile compound was quantified by comparing its ion peak area with
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internal standard 4-octanol. The concentration was calculated on a dry basis by considering the
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moisture content and using the following equation according to Xiao, Lee, Zhang, Ebeler,
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Wickramasinghe, Seiber, et al. (2014) as follows:
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µ
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ℎ 1 µ = × . . . ×
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2.5. Statistical analysis
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The sorghum tea processing was repeated three times, and the analysis of phenolic content and
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antioxidant activity was done in triplicates on each repeated sample (a total of 9 replicates). The
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analysis of moisture contents and volatile compounds was done on each repeated sample (a total of 9
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3 replicates). The significant difference of means between each processing step was determined
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using one-way ANOVA with Turkey grouping at 95% confidence level (Minitab Express, version
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1.2.0, Sydney, Australia). Due to the large amount of background noise and the impurity peak in the
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chromatograms of grain infusion sample, an additional one-way ANOVA test was performed for
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the data of total aldehyde and total pyrazine with the grain infusion sample data removed.
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231 3. Results and Discussion
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3.1. Phenolic contents
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It was reported that soaking could alter the physical structure and functionality of grains (Ebuehi &
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Oyewole, 2008). Prolonged soaking increases the water content and leads to rupture of grain cells,
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releasing the cellular contents, which further accelerates the leaching of water-soluble compounds
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(Ituen, Mittal, & Adeoti, 1986; Wu et al., 2013). However, in the present study, the soaking process
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increased the TFC in sorghum while TPC and CTC were not affected (Table 1). A possible
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explanation for this might be that the white sorghum variety (Liberty) used in this study had
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relatively lower phenolic contents as compared to other sorghum genotypes such as red and brown
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sorghums (Wu et al., 2017), and most of the phenolic compounds in sorghum are in the bound form
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(Dykes, Rooney, Waniska, & Rooney, 2005; Wu et al., 2017). Minor leaching of phenolic
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compounds might have occurred during the soaking process, but the loss of TPC and CTC might be
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negligible to cause statistical significant differences. The slight increase of TFC might be due to the
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release of some bound phenolic compounds when the water penetrated into cell walls, as the above
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method (Section 2.3.2) were mainly able to extract free phenolic compounds (Wu et al., 2013).
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Steaming can severely damage the cellular structure of grains, which may cause the release of some
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bound phenolic compounds (Randhir, Kwon, & Shetty, 2008; Wu et al., 2013). In addition, the heat
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effect during the steaming process can also lead to the degradation of some phenolic compounds
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such as vanillic and p-coumaric acids (Wu et al., 2013). Interestingly, our results showed that the 10
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steaming process increased (P ≤ 0.05) TFC in sorghum but had no significant (P > 0.05) effect on
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TPC and CTC (Table 1). The reason could be similar to the above soaking process that there was a
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relatively lower phenolic content in this sorghum variety. Some phenolic compounds might be
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damaged during the process, while some bound phenolic compounds might be released, so the
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overall phenolic compounds remained unchanged. Wu et al. (2013) also reported elevated free
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ferulic acid and bound p-coumaric acid levels in the steamed red sorghum grain.
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The final sorghum grain tea product was obtained after the roasting process, which slightly
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increased the TPC and CTC, while TFC (P > 0.05) was significantly enhanced compared to the
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steamed samples (Table 1). In comparison with the initial raw sorghum, the TPC, TFC and CTC in
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roasted sorghum were all significantly (P ≤ 0.05) higher. The result is consistent with that of red
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sorghum grain tea processing as reported by Wu et al. (2013). Two mechanisms have been
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proposed: (1) roasting can cause damage to cell wall and break down cellular constituents, therefore,
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releases some bound phenolic compounds (Randhir, Kwon, & Shetty, 2008); (2) at high
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temperature, some high molecular weight or polymeric phenolic compounds such as condensed
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tannins are break down into simple and extractable molecules (Cheng, Su, Moore, Zhou, Luther,
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Yin, et al., 2006).
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3.2. Antioxidant activity
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It is widely recognised that the grain phenolic compounds are the main contributors to antioxidant
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activity (de Morais Cardoso, Pinheiro, Martino, & Pinheiro-Sant'Ana, 2017; Dykes & Rooney,
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2006; Stefoska-Needham, Beck, Johnson, & Tapsell, 2015). The ABTS and DPPH radical
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scavenging activity of the extracts from raw and processed sorghum grains are shown in Table 1,
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which suggests that the soaking, steaming and roasting process did not affect (P > 0.05) the ABTS
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and DPPH radical scavenging activity. Although the above results indicated that the TPC, TFC and
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CTC in the roasted sorghum were significantly (P ≤ 0.05) higher than the raw sorghum, the
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antioxidant activity remained unchanged, implying that the relatively low phenolic compounds in
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this white sorghum variety might have less impact on its antioxidant activity compared to coloured
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sorghum, other compounds might also contribute to the antioxidant activity. It was reported that
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vitamins, peptides and proteins also have strong antioxidant activities (Elias, Kellerby, & Decker,
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2008; Gliszczyńska-Świgło, 2006), and their contributions to the antioxidant activity of the present
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sorghum variety need further investigation.
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284 3.3. Sorghum volatile profile
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The volatile compounds of raw and processed sorghum grain and sorghum grain tea infusions are
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summarised in Table 2, with representative GC-MS chromatogram of raw sorghum and final
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sorghum tea product (roasted sorghum) presented in Fig. 2. This represents the very first research
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on sorghum tea volatile profiling. Since the volatile profiles were measured in different forms of
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products, including solid raw sorghum, intermedium processed products and liquid tea product, it is
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unlikely to construct chemical standard calibration curves to accurately simulate each product
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condition. Semi-quantification was used in this study for the profiling of sorghum derived volatiles,
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which could effectively achieve the purpose of the current study to investigate the influences of
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processing technology on sorghum volatile profile. A total of 63 volatile compounds were detected
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from the sorghum samples. Amongst those compounds, 45 were identified based on mass spectra
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(MS) and retention index (RI) from the in-house NIST library 11.0 and NIST webbook (NIST,
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2017), while 18 were unidentified. The identified volatile compounds were classified into eight
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chemical groups including 5 alcohols, 13 alkanes, 2 aldehydes, 2 carboxylic acids, 15 esters, 4
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ketones, 3 pyrazines and 1 phenylenediamine. The unidentified compounds had distinct but weak
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signals (Fig. 2) and unidentifiable by the NIST (11.0) MS database. However, the MS information
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indicated that these compounds were most likely to be 10-12 carbon alkanes or its derivatives.
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3.4. Raw sorghum volatile compound profile
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3.4.1. Esters
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Esters were the dominant volatile compounds in raw sorghum, and the total ester content was 33.83
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± 1.35 µg/g db (Table 2). Amongst the 15 detected esters, methyl esters were the dominant ester in
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tested sorghum. These esters contributes to fruity, green, sweet, and floral aroma and flavour of
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food and beverages (Acree & Arn, 2017; TGSC, 2017).
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3.4.2. Alkanes
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Alkanes were the second most abundant volatile compounds in the tested sorghum (Table 2).
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Thirteen alkanes were detected in raw sorghum, and the total alkane content was 15.28 ± 0.61 µg/g
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db. In general, alkanes have waxy and gasoline-like odour (Acree & Arn, 2017; TGSC, 2017).
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Although the alkane content in sorghum was high, it might not make a significant contribution to
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aroma due to their low odour intensity and high odour threshold values (He, Guo, Yang, Xie, Ju, &
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Guo, 2016; Zanan et al., 2016). Alkanes, aldehydes and alkenes are associated with lipid oxidation
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products, and high concentrations of these compounds may cause off-flavour in food
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products(Bryant & McClung, 2011).
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3.4.3. Alcohols
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Alcohols were the third most abundant volatile compounds (13.31 ± 0.61 µg/g db) in sorghum
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(Table 2). Five alcohols including 1-hexanol (1), 1-octanol (13), 1,6-octadien-3-ol, 3,7-dimethyl-
323
(17), phenylethyl alcohol (19) and 1-nonanol (25) were detected, with a noticeable high
324
concentration (8.95 ± 0.44 µg/g db) of 1-hexanol. These alcohols are associated with intense sweet,
325
floral, waxy, green, citrus and fruity odour descriptions (Acree & Arn, 2017; TGSC, 2017).
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3.4.4. Other compounds
328
Other compounds including aldehydes, carboxylic acids and ketones were detected in small
329
quantities in sorghum (Table 2). Aldehydes of nonanal (18) and 2,4-decadienal (47) were
330
determined, in which nonanal (18) has waxy, aldehydic and citrus odour, and 2,4-decadienal (47)
331
has fatty, oily and chicken skin-like odour (TGSC, 2017). The carboxylic acid of hexanoic acid (5)
332
is responsible for fatty, sweat and cheese odour, and the ketones of 2(3H)-furanone, 5-ethyldihydro-
333
(10) and 2(3H)-furanone, dihydro-5-pentyl- (50) have pleasant sweet and creamy-like aroma
334
(TGSC, 2017).
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335
3.5. Effect of soaking processing on the sorghum volatile compounds
337
The purpose of soaking was to increase the grain moisture content, swell and soften the physical
338
structure, thus to improve the subsequent thermal treatment efficiency. Surprisingly, soaking had a
339
great impact (P ≤ 0.05) on the volatile compounds (Table 2). The TVC (161.72 ± 26.42 µg/g db)
340
was increased about two folds compared to the raw sorghum, which was mainly contributed by the
341
ester compounds as the total ester content (122.16 ± 27.25 µg/g db) increased approximately four
342
folds compared to the raw grain. It was suggested that soaking can lead to a high cell stress and
343
consequent rupture and thus result in more efficient extraction of esters (Ituen, Mittal, & Adeoti,
344
1986; Wu, et al., 2013). The enzymatic esterification reactions of short chain acids and alcohols
345
might have also contributed to the increased esters during the soaking process because sufficient
346
water content could activate the lipase activity to produce esters (Gubicza, Kabiri-Badr, Keoves, &
347
Belafi-Bako, 2000; Wehtje, Kaur, Adlercreutz, Chand, & Mattiasson, 1997).
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349
Alkanes were the second most abundant volatile compounds in soaked sorghum grains. The total
350
alkane content was 17.74 ± 1.69 µg/g db, which was higher than the raw sorghum (P ≤ 0.05).
351
Decane, 4-methyl- (9), decane, 3,7-dimethyl- (11), undecane, 2,6-dimethyl- (30), tridecane (46) and
352
pentadecane (59) were the most abundant alkanes in soaked sorghum. A significant (P ≤ 0.05) 14
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increase of aldehydes, especially nonanal (18), was also observed in the soaked sorghum compared
354
to the raw seed. Alkanes are a type of plant lipid and water-insoluble, which are mainly located in
355
the germ of grains and can be difficult to extract (Cheek & Jebb, 2001; Waniska & Rooney, 2000).
356
The results indicated that soaking sorghum for 17 h could increase the extraction efficiency of
357
alkanes as well as the aldehydes.
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The alcohol content (5.14 ± 0.15 µg/g db) was significantly (P ≤ 0.05) reduced in the soaked
360
sorghum compared to the raw (Table 2). In addition, carboxylic acids eg. hexanoic acid (5), which
361
were relatively high in raw sorghum but were not detected in the soaked sorghum. Similarly, ketone
362
levels were also greatly lowered, and 2(3H)-furanone, 5-ethyldihydro- (10) was not detected in the
363
soaked sorghum. The reason could be that alcohols, carboxylic acids and ketones especially small
364
molecular weight (MW) molecules such as 1-hexanol (1) and hexanoic acid (5) are water-soluble,
365
and may have been leached out during the soaking process (Lucas, Le Ray, & Mariette, 2007). The
366
reduction of alcohol content also partly supported the postulation that they might have been
367
involved in esterification reactions.
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3.6. Effect of steaming processing on the sorghum volatile compounds
370
The steaming process dramatically reduced the TVC (Table 2) in contrast to the soaking process.
371
The TVC in steamed sorghum was 52.71 ± 4.60 µg/g db which was about three folds lower than in
372
the steamed grain, with this reduction being mainly due to the loss of ester content. After the
373
steaming process, some esters found in soaked grain were not detected, and the most abundant ester
374
compounds (i.e. peak 62, 63 and 64) that were previously found in raw and soaked sorghum were
375
barely detectable. The alcohol content was also reduced by the streaming process, and phenylethyl
376
alcohol (19) found in the raw and soaked grains was not detected in the steamed sorghum. A
377
possible explanation for this might be the decomposition of esters and leaching of alcohols. Esters
378
are only very slightly soluble in water and might not be affected by leaching, but they could have
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379
evaporated from the sample during the steaming process or have been hydrolysed into fatty acids
380
and alcohols (Rodrigues & Vale, 2009).
381 The alkane and aldehyde contents of the grains were further increased (P ≤ 0.05) upon steaming.
383
Alkane was the most abundant volatile compound in the steamed sorghum, with a total
384
concentration of 23.03 ± 2.41 µg/g db, and aldehyde had a total concentration of 3.50 ± 0.09 µg/g
385
db. The steaming process can damage the cellular structure of the grains, and thus might have led to
386
the increased extractability (Randhir, Kwon, & Shetty, 2008; Wu et al., 2013). This may also have
387
facilitated lipid oxidation thus more production of alkane and aldehyde (Bryant and McClung,
388
2011). Another reason of high abundance of alkane in steamed sorghum might be that these
389
alkanes were mostly 10-12 carbon and are relatively stable during wet heat processing.
390
Nevertheless, the carboxylic acid and ketone contents were not affected by the steaming process,
391
indicating they were stable under the steaming conditions used.
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3.7. Effect of roasting processing on the sorghum volatile compounds
394
The roasted sorghum is the final sorghum tea product ready for infusion. Although the roasting
395
process had no significant (P > 0.05) effect on the TVC (47.26 ± 4.42 µg/g db), the profile of
396
volatile compounds was changed (Table 2). For example, pyrazines were only detected after the
397
roasting process. The total pyrazine content in the roasted sorghum was 3.06 ± 0.29 µg/g db, which
398
included pyrazine 2,5-dimethyl- (2), pyrazine, 2-ethyl-3, 5-dimethyl- (14) and pyrazine,
399
tetramethyl- (15). These pyrazines generally have roasted and nutty odour attributes (TGSC, 2017).
400
It has been suggested that pyrazines can be biosynthesized by microorganisms such as Bacillus
401
cereus or by heating processing (Adams & De Kimpe, 2007; Guo, et al., 2017). During the heating
402
process, pyrazines are formed by Maillard reactions of amino acids with reducing sugars and
403
Strecker degradations (Alasalvar, Shahidi, & Cadwallader, 2003). In addition, 1,2-benzenediamine,
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404
4-methyl- (7) was also only found in the roasted sorghum, and this compound is likely to be
405
produced via a similar mechanism as pyrazine and possibly contributed to the roasted aroma.
406 Other volatile compounds such as alcohol, alkane, aldehyde, ester, carboxylic acid and ketone
408
contents remained relatively stable during the roasting process. However, compared to the raw
409
sorghum, the aldehyde and alkane contents in the roasted sorghum were slightly higher, while the
410
alcohol, ester, carboxylic acid and ketone contents were significantly lower, and many of them were
411
not detected in the roasted sorghum (Table 2). Processing has a great impact on the volatile profile
412
of sorghum grain and the mechanism needs further investigation.
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415
The volatile compounds in the roasted sorghum whole grain infusion (GI) and grain powder
416
infusion (PI) were compared (Table 2). The TVC in PI was significantly higher (P>0.05) than in GI,
417
and more individual volatile compounds were detected in the PI tea. The results suggest that the
418
extraction of volatile compounds in the powder form infusion tea was more effective than the whole
419
grain form infusion tea. Therefore, the powder form of roasted sorghum grain could be more
420
suitable for sorghum tea making. In addition, this roasted sorghum grain powder could be further
421
processed into consumer-friendly forms such as tea bags. However, it should be noted that no
422
significant (P > 0.05) difference in TVC was observed between these two type teas (Table 2). This
423
was due to the significant data variation, which was caused by the interference of background noise
424
and impurity peaks in the chromatograms of the infusion samples, and these impurity peaks were
425
identified as plastic materials and might come from the headspace vial and cap materials under the
426
prolonged infusion of boiling water. A methodology development, such as by varying extraction
427
temperature, time and fibre type, is required to optimise the characterisation of infusion volatiles in
428
the future study.
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429 17
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Regardless of the form of grain used for infusion, a significant increase of the pyrazine and
431
phenylenediamine contents was observed upon infusion as compared to the roasted sorghum grain.
432
The increase of the pyrazine contents might be due to the fact that pyrazine is water soluble, and
433
infusion with boiling water might cause swelling of grains and thus releasing pyrazine from the
434
matrix (Ituen, Mittal, & Adeoti, 1986; Wu et al., 2013). Another possible explanation is that heating
435
by boiling water during the infusion could lead to Maillard reaction and thus generate more
436
pyrazines (Morini & Maga, 1995).
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SC
437 4. Conclusion
439
In this study, a sorghum grain tea was developed using a white sorghum variety (Liberty) through
440
three consecutive processes of soaking, steaming and roasting. Compared to the raw sorghum grain,
441
the phenolic contents (TPC, TFC, CTC) in the roasted sorghum were significantly higher, although
442
antioxidant activity (DPPH, ABTS) was not affected, suggesting other compounds might have
443
contributed to the antioxidant activity of the grains. In terms of the volatile compounds, a total of 63
444
volatile compounds including 5 alcohols, 13 alkanes, 2 aldehydes, 2 carboxylic acids, 15 esters, 4
445
ketones, 3 pyrazines and 1 phenylenediamine were detected in the sorghum samples. The soaking
446
process dramatically increased the ester content as well as alkane, aldehyde, while reduced the
447
alcohol, carboxylic acids and ketone contents. Steaming, however, significantly reduced the ester
448
and alcohol contents, while increased the alkane and aldehyde contents. The roasting process
449
resulted in the formation of pyrazine and phenylenediamine compounds. In addition, powder form
450
infusion was more effective in extracting volatile compounds than whole grain form infusion,
451
therefore the powder form may be more suitable for sorghum tea making. According to the detected
452
volatile compounds, the white sorghum tea may have an overall floral, sweet, waxy, and nutty
453
aroma. Future studies are required to compare the effect of processing on different sorghum
454
varieties (e.g. red and black sorghums which have higher phenolic contents), as well as sensory
455
evaluation of the infused sorghum grain tea products.
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456 457
5. Conflict of interest
458
None.
459 6. References
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Wu, G., Johnson, S. K., Bornman, J. F., Bennett, S. J., & Fang, Z. (2017). Changes in whole grain
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Wu, G., Johnson, S. K., Bornman, J. F., Bennett, S. J., Singh, V., Simic, A., & Fang, Z. (2016). Effects of
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genotype and growth temperature on the contents of tannin, phytate and in vitro iron availability of
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Figure captions
554
Fig. 1. Flow diagram of sorghum grain tea processing, sampling and analysis.
555
Fig. 2. GC-MS chromatograms showing the volatile compound profile of raw (a) and roasted (b)
556
sorghum. Peak numbers are identified in Table 2; peak No. 6 is the internal standard (I.S.) 4-octanol.
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Fig. 1 Flow diagram of sorghum grain tea processing, sampling and analysis.
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Fig. 2. GC-MS chromatograms showing the volatile compound profile of raw (a) and roasted (b)
561
sorghum. Peak numbers are identified in Table 2; peak No. 6 is the internal standard (I.S.) 4-octanol.
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Table 1: Effect of processing on TPC, TFC, CTC, and ABTS and DPPH radical scavenging activity
563
of sorghum grain. Analysis
Processing Soaked
Steamed
Roasted
TPC
0.34 ± 0.02b
0.33 ± 0.02b
0.36 ± 0.02ab
0.41 ± 0.03a
TFC
0.71 ± 0.02d
0.78 ± 0.02c
0.89 ± 0.03b
0.94 ± 0.03a
CTC
3.26 ± 0.18b
3.13 ± 0.20b
3.44 ± 0.18ab
3.89 ± 0.33a
ABTS
2.01 ± 0.15a
1.95 ± 0.09a
1.94 ± 0.20a
2.14 ± 0.09a
DPPH
1.42 ± 0.07a
1.39 ± 0.10a
1.37 ± 0.09a
1.49 ± 0.15a
SC
RI PT
Raw
TPC = total phenolic content (mg GAE/g db).
565
TFC = total flavonoid content (mg CAE/g db).
566
CTC = condensed tannin content (mg CAE/g db).
567
ABTS and DPPH = ABTS and DPPH radical scavenging activity (mg TE/g db).
568
a, b, c
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= values with different superscripts in the same row are significantly different (P ≤ 0.05).
26
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569
Table 2: Effect of processing and infusion on sorghum grain tea volatile compounds (µg/g sample
570
db). Pea
Compound Name
RI*
k
Processing Raw
Soaked
Steamed
Roasted
No. 1
1-Hexanol
867
8.95±0.44
a
2.70±0.17
b
0.82±0.10
c
ND
Powder
Grain
infusion
infusion
ND
ND
ID**
Aroma***
RI,
Pungent, ethereal, fruity
MS 1-Octanol
107
0.60±0.03a
0.48±0.07a
0.19±0.02b
0.18±0.01b
0.19±0.02b
0 17
19
1,6-Octadien-3-ol, 3,7-
109
dimethyl-
9
Phenylethyl Alcohol
110
2.17±0.14a
1.52±0.10ab
2.21±0.17a
1.75±0.19ab
1.8±0.21a
1.08±0.54b
117
0.73±0.04a
0.19±0.02b
ND
ND
ND
ND
0.86±0.07a
0.25±0.02b
13.31±0.61
102
0.14±0.02bc
5.14±0.15
b
3.36±0.09
0.97±0.04b
1.40±0.12a
c
1.77±0.20a
2 11
Decane, 3,7-dimethyl-
105
Dodecane, 2,6,10-
106
trimethyl-
0
23
Undecane, 2-methyl-
116
28
Dodecane
120
29
Undecane, 2,4-
0.12±0.02c
2.05±0.21
d
dimethyl-
0
30
Undecane, 2,6-
121
dimethyl-
3
Undecane, 4,8-
122
dimethyl-
2
Dodecane, 4,6-
125
dimethyl-
2
46
Tridecane
129
RI,
2.23±0.20
cd
1.68±0.24a
1.47±0.73
Aldehydic, waxy, citrus, tart,
MS
sweet
RI,
--
d
ND
MS
2.82±0.40a
2.84±0.41a
0.32±0.17c
MS
--
0.72±0.03b
1.03±0.09a
1.29±0.14a
1.14±0.13a
1.16±0.18a
0.14±0.07c
MS
--
0.52±0.05c
0.55±0.06bc
0.81±0.13a
0.71±0.10ab
0.66±0.10abc
ND
RI,
--
1.09±0.07b
1.16±0.15ab
1.51±0.17a
1.49±0.17a
1.30±0.16ab
0.37±0.17c
MS
Alkane
--
TE D
0.36±0.02c
MS
0.39±0.07bc
0.53±0.06a
0.47±0.05ab
0.38±0.03c
ND
RI,
1.34±0.25ab
1.83±0.32a
1.64±0.28a
1.35±0.10ab
0.13±0.05c
MS
--
0.77±0.10ab
1.00±0.14a
0.95±0.13a
0.75±0.11ab
ND
MS
--
0.90±0.13ab
1.08±0.16ab
1.04±0.14a
0.72±0.09c
ND
MS
--
4.79±0.11b
4.79±0.22b
7.97±0.72a
6.79±0.19a
5.57±0.70b
0.40±0.20c
MS
Gasoline-like to odourless
0.76±0.07ab
0.89±0.12ab
0.97±0.12a
0.76±0.08bc
0.54±0.04c
0.24±0.09d
MS
Gasoline-like, mild waxy
0.76±0.08b
0.84±0.24a
0.43±0.05c
0.37±0.09cd
0.20±0.10d
ND
MS
Waxy
0.59±0.05c
1.16±0.13a
0.75±0.00b
0.40±0.04d
0.24±0.04d
ND
MS
Waxy
15.28±0.61c
17.74±1.69ab
23.03±2.41
20.16±1.89a
17.39±2.29bc
1.60±0.74d
a
b
RI,
Waxy, aldehydic, citrus
1.27±0.11b
EP
0.68±0.08b
0.85±0.07bc
AC C
36
0.15±0.10bc
3.08±0.30a
0
31
1.59±0.23a
0.15±0.01bc
2.51±0.22a
4
121
Sweet, floral, fresh, bready
1.92±0.16b
4 12
RI,
M AN U
Decane, 4-methyl-
Citrus, orange, floral, waxy, rose
MS
a
9
Waxy, green, citrus, floral
MS
1 Total alcohol
RI,
SC
1-Nonanol
RI, MS
8 25
0.24±0.13b
RI PT
13
MS
9
54
Tetradecane
139
9
58
Heneicosane
148
5
59
Pentadecane
149 9
Total alkane
18
Nonanal
110
47
2,4-Decadienal
131
0.54±0.03b
1.67±0.11ab
2.62±0.13ab
1.39±0.14ab
2.76±0.10ab
3.66±2.26a
0.04±0.01a
0.13±0.01a
0.87±0.05a
0.72±0.14a
2.12±0.17a
5.98±6.99a
0.58±0.03a
1.80±0.11aC
3.50±0.18aB
2.12±0.28aC
4.88±0.07aA
9.64±9.25a
3
MS
MS
5 Total aldehyde
RI,
27
Fatty, oily, chicken skin-like
ACCEPTED MANUSCRIPT
D
5
Hexanoic acid
979
2.09±0.52
ND
ND
ND
ND
ND
RI,
Fatty, sweat, cheese
MS 37
0.78±0.04c
0.89±0.04bc
1.24±0.11a
0.99±0.10ab
0.96±0.07bc
0.18±0.12d
2.87±0.51a
0.89±0.04b
1.24±0.11b
0.99±0.10b
0.96±0.07b
0.18±0.12c
924
7.83±0.12a
6.92±0.93a
ND
ND
ND
ND
109
4.73±0.40a
4.19±0.38a
0.45±0.12b
ND
ND
ND
1.41±0.03b
2.21±0.29a
ND
ND
ND
ND
3-Thiophenecarboxylic
125
acid
7
Total carboxylic acid 3
Hexanoic acid, methyl
16
Benzoic acid, methyl ester
0
20
Octanoic acid, methyl
112
ester
3
Methyl nicotinate
113
ester
7
Nonanoic acid, methyl
122
ester
3
Cyclopentanecarboxyli
122
c acid, 2-pentyl ester
8
Butanoic acid, hexyl
136
ester
5
53
Hexanoic acid, hexyl
138
ester
3
55
Benzoic acid, 2-
143
52
methylbutyl ester
2
60
Dodecanoic acid,
152
methyl ester
2
61
Pentanoic acid, 2,2,4-
158
trimethyl-3-
2
0.73±0.06b
1.08±0.04a
0.34±0.02c
ND
ND
ND
ND
ND
1.14±0.09c
1.21±0.03bc
1.75±0.14a
1.33±0.12abc
1.59±0.09ab
0.77±0.03a
0.71±0.10a
0.65±0.23ab
0.34±0.04bc
0.32±0.03bc
0.19±0.01a
ND
0.12±0.01b
0.10±0.02bc
ND
ND
0.81±0.06a
0.49±0.05b
1.96±0.23a
0.84±0.07b
0.93±0.30a
b
9,12-Octadecadienoic
209
acid (Z,Z)-, methyl
1
73.89±17.38a
EP
2
1.58±0.11b
AC C
1.63±0.07b
16.08±5.25a
9.09±2.51a
1.27±0.92a
0.51±0.33d
MS
--
0.27±0.07c
MS
Green, waxy, soapy and fruity
0.07±0.01c
ND
RI,
Green, sweet, fruity, tropical
0.52±0.05b
0.69±0.11ab
0.18±0.11c
MS
--
ND
ND
ND
ND
RI,
Oily, wine, fruity, floral
0.57±0.06c
0.33±0.10c
0.26±0.06c
0.53±0.10bc
0.41±0.09b
0.18±0.07b
0.23±0.03b
0.05±0.00b
ND
ND
ND
ND
0.05±0.00b
ND
ND
6.60±0.27bc
3.67±0.26bc
4.21±0.60bc
2.75±1.54c
0.234±0.11
33.83±1.35
122.16±27.25
b
a
ND
ND
ND
10
2(3H)-Furanone, 5-
104
0.80±0.05
ND
ND
ND
ND
ND
ethyldihydro-
7
2-Decanone
119
2
Pyrazine, 2,5-dimethyl-
913
--
RI,
--
RI,
Odourless
MS
--
MS RI,
Sweet, creamy, lactonic, tobacco
MS 0.11±0.01cd
0.10±0.01d
0.15±0.01cd
0.94±0.17bc
1.46±0.07ab
2.25±0.73a
1
3
RI,
MS
ND
Total ketone
MS
MS
0.49±0.03a
dihydro-5-pentyl-
MS
MS
0.47±0.07a
135
Sweet, wintergreen, fruity
Sweet, fruity, waxy, tropical, wine
971
2(3H)-Furanone,
RI,
MS
4-Octanone
50
Warm, herbal, tobacco
ND
4
27
RI,
b
7
Total ester
1.27±0.20a
ND
methyl-, methyl ester
methyl ester, (E)-
0.73±0.15ab
3.61±0.33a
10.03±0.36
209
1.18±0.14ab
2.45±0.13b
192
9-Octadecenoic acid,
Green, fruity, waxy, citrus, fatty
MS
Pentadecanoic acid, 14-
ester 64
0.29±0.12ab
ND
isobutyl ester
63
Phenolic, cherry
MS
carboxyisopropyl,
62
RI,
SC
ester
RI,
MS
M AN U
116
33
Fruity, pineapple, ether
MS
TE D
Benzoic acid, ethyl
32
RI, MS
3 24
--
RI PT
21
MS
RI,
Orange, floral, fatty, peach
MS 2.98±0.12a
1.05±0.09b
0.89±0.08b
0.51±0.11b
0.61±0.11b
0.80±0.44b
4.36±0.21a
1.64±0.11c
1.04±0.08c
1.44±0.20c
2.07±0.04bc
3.05±1.17ab
ND
ND
ND
2.04±0.18b
4.95±0.10ab
6.57±3.43a
RI,
Sweet, coconut, coumarin, creamy
MS
RI,
Roasted, Nutty, peanut, musty
MS 14
Pyrazine, 2-ethyl-3,5-
107
ND
ND
ND
0.91±0.12b
28
6.48±0.07a
7.88±4.45a
RI,
Roasted, nutty
15
dimethyl-
3
Pyrazine, tetramethyl-
108
ACCEPTED MANUSCRIPT
MS
ND
ND
ND
0.11±0.00b
0.70±0.18ab
1.05±0.59a
ND
ND
ND
3.06±0.29bB
12.13±0.28ab
15.50±8.46a
RI,
Roasted
MS
0 Total pyrazine
A
7
1,2-Benzenediamine, 4-
100
methyl-
2
Total
ND
ND
ND
1.28±0.08b
5.83±0.15a
7.45±3.72a
ND
ND
ND
1.28±0.08b
5.83±0.15a
7.45±3.72a
11.07±0.73
12.34±1.40a
13.95±1.62
12.49±1.86a
9.84±1.53a
2.61±1.76b
47.26±4.42b
59.53±4.42b
44.25±27.43
MS
--
a
Total
a
81.29±3.25
161.72±26.42
52.71±4.60
b
a
b
b
RI PT
phenylenediamine Total unknown ****
* RI = retention index
572
** ID = Identification by comparison of mass spectra (MS) with in-house NIST library 11.0, and
573
MS and retention index (RI) of NIST Webbook (NIST, 2017).
574
*** = Aroma descriptions were obtained from Acree and Arn (2017) and TGSC (2017)
575
**** = Sum of all the unidentifiable compounds (peak No. 8, 22, 26, 34, 35, 38-45, 48, 49, 51, 56,
576
57).
577
ND = Not detected or value below the detection threshold.
578
-- = Not available.
579
a-f
580
A-C
581
and values with different superscripts in the same row are significantly different (P ≤ 0.5)
TE D
M AN U
SC
571
= Values with different superscripts in the same row are significantly different (P ≤ 0.05).
AC C
EP
= Additional one-way ANOVA test was performed to remove the large error grain infusion data,
29
ACCEPTED MANUSCRIPT
Highlight: Sorghum grain was soaked, steamed, and roasted to make grain tea.
•
Processing techniques affected total phenolic content but not antioxidant activity.
•
A total of 63 volatile compounds were detected in sorghum grain tea.
•
Main volatile compounds were alcohols, alkanes, aldehydes, and esters.
•
More volatile compounds were extracted in sorghum powder than whole grain infusion.
AC C
EP
TE D
M AN U
SC
RI PT
•
S0733-5210(18)30614-3
DOI:
https://doi.org/10.1016/j.jcs.2018.10.012
Reference:
YJCRS 2659
To appear in:
Journal of Cereal Science
Received Date: 9 August 2018 Revised Date:
19 September 2018
Accepted Date: 31 October 2018
Please cite this article as: Xiong, Y., Zhang, P., Luo, J., Johnson, S., Fang, Z., Effect of processing on the phenolic contents, antioxidant activity and volatile compounds of sorghum grain tea, Journal of Cereal Science (2018), doi: https://doi.org/10.1016/j.jcs.2018.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT Effect of processing on the phenolic contents, antioxidant activity and volatile
2
compounds of sorghum grain tea
3
Yun Xiong1, Pangzhen Zhang1, Jiaqian Luo1, Stuart Johnson2, Zhongxiang Fang1*
4 5
1
6
Melbourne, Parkville, VIC 3010, Australia
7
2
8
University, Perth, WA 6845, Australia
RI PT
School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, University of
9
SC
School of Molecular and Life Sciences, Curtin Health Innovation Research Institute, Curtin
*Corresponding author: Dr Zhongxiang Fang
11
School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, University of
12
Melbourne, Parkville, VIC 3010, Australia
13
Email: [email protected]; Tel: +61 3 83445063
AC C
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10
ACCEPTED MANUSCRIPT
Abstract: Sorghum grain is rich in phenolic compounds and may be used to develop functional tea
15
beverages. This work investigated the effect of processing techniques on the phenolic contents,
16
antioxidant activity, and volatile compounds of a white colour sorghum (Liberty) grain tea.
17
Significant (P ≤ 0.05) increase of total phenolic content, total flavonoid content and condensed
18
tannin content were observed during the processing, whereas the antioxidant activity was not
19
statistically enhanced. A total of 63 volatile compounds were detected including 5 alcohols, 13
20
alkanes, 2 aldehydes, 2 carboxylic acids, 15 esters, 4 ketones, 3 pyrazines and 1 phenylenediamine,
21
which were affected by the processing techniques. The sorghum tea made from powder form
22
infusion had more abundant volatile compounds compared to whole grain form infusion. The
23
findings of this research have potential to expand human consumption of sorghum grain in the new
24
form of grain tea.
25
Keywords: Sorghum grain tea; processing technology; phenolic content; volatile compounds.
AC C
EP
TE D
M AN U
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14
1
ACCEPTED MANUSCRIPT
1. Introduction
27
Sorghum (Sorghum bicolor L. Moench) is the fifth leading cereal crop in the world (Wu et al.,
28
2013). It contains high levels of bioactive phytochemicals, particularly phenolic compounds such as
29
phenolic acids, flavonoids and condensed tannins (Awika & Rooney, 2004; de Morais Cardoso et
30
al., 2017). Studies have shown that sorghum grain has high antioxidant activity, good cholesterol-
31
lowering ability, anti-inflammatory and anti-carcinogenic properties (de Morais Cardoso et al.,
32
2017; Dykes & Rooney, 2006; Stefoska-Needham et al., 2015). It is suggested that regular
33
consumption of sorghum grain have the potential of reducing the risk of cancers, cardiovascular
34
diseases and type II diabetes (Awika & Rooney, 2004; Okarter & Liu, 2010).Therefore, there is an
35
increased interest using sorghum grain to develop functional foods. Recently, application of cereal
36
grain to make grain tea beverage is a new trend. For example, Tartary buckwheat, barley and brown
37
rice tea have been developed as functional tea beverage owing to their unique flavour, taste and
38
health-promoting benefits (Guo et al., 2017; Yuki, 2017). Sorghum grain could be an idea material
39
to be developed into grain tea. The additional advantage of using sorghum grain as tea making
40
material is that sorghum is a sustainable grain that could be grown in a semi-arid environment
41
(Stefoska-Needham et al., 2015) and the cost of sorghum grain is much lower compared to leaf tea.
42
However, limited research has been conducted on sorghum grain tea processing with only one paper
43
investigating the phenolic compositions and biological activities of a red sorghum grain tea (Wu, et
44
al., 2013).
SC
M AN U
TE D
EP
AC C
45
RI PT
26
46
Consumers’ satisfaction of tea is affected by its quality, in which aroma is a key quality indicator
47
(Kraujalytė, Pelvan, & Alasalvar, 2016). Therefore, it is critical for the food industry to understand
48
the aroma profile of sorghum grain tea and how it is affected by the processing technology. The
49
aroma profile of raw sorghum grain has been recently reported by Zanan, Khandagale, Hinge,
50
Elangovan, Henry, and Nadaf (2016), where a total of 29 volatile compounds were detected in two
51
sorghum varieties of E228 and M35-1. In another research, Chughtai, Pasha, Anjum, and Nasir 2
ACCEPTED MANUSCRIPT
(2015) detected a total of 35 volatile compounds from seven commercial sorghum varieties.
53
However, no information is currently available about the volatile profile of sorghum grain tea.
54
Therefore, the objective of this study was to investigate the effect of sorghum grain tea processing
55
techniques (soaking, steaming and roasting) on the phenolic contents, antioxidant activity and
56
aroma profile. To our knowledge, this research is the first to report volatile profiles of sorghum
57
grain tea product and the influences of processing techniques on volatile composition.
RI PT
52
58 2. Materials and Methods
60
2.1. Processing of sorghum grain tea
61
A non-tannin white colour sorghum variety (Liberty) was supplied by Nuseed (Queensland,
62
Australia). The selection of this variety is because it is the main commercial sorghum for human
63
consumption in Australia. The sorghum grain tea was produced through three consecutive processes:
64
soaking, steaming and roasting, as described by Wu et al. (2013) with some modifications (Fig. 1).
65
The steaming and roasting processes were both carried out on a commercial oven (Convotherm 4
66
easy Dial 10.10 multifunction oven, Wolfratshausen, Germany). During the process, raw, soaked,
67
steamed and roasted sorghum grains were sampled and stored in the dark at –20 °C until analysis.
68
EP
TE D
M AN U
SC
59
2.1.1. Soaking
70
About 200 g raw whole sorghum grain was used in each replicate and 3 replicates were conducted.
71
The raw sorghum was soaked in water (1:4 w/v) for 17 h at room temperature. The soaked sorghum
72
grain was washed, drained, and 50 g of the soaked sample were collected for analysis, with the
73
remaining sent for steaming process.
AC C
69
74 75
3
ACCEPTED MANUSCRIPT
76
2.1.2. Steaming
77
About 100 g of the soaked sorghum grain was steamed at 100 °C for 50 min, then about 50 g of the
78
steamed sorghum grain was cooled and collected for analysis, and the remaining was sent for
79
roasting process.
RI PT
80 2.1.3. Roasting
82
About 50 g of the steamed sorghum grain was roasted at 150 °C for 60 min, cooled, collected and
83
subjected to analysis.
SC
81
84 2.2. Chemicals and materials
86
Folin & Ciocalteu phenol reagent, gallic acid, aluminium chloride, (+)-catechin hydrate, vanillin,
87
2,2-Diphenyl-1-picrylhydrazyl (DPPH), (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
88
acid (Trolox), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),
89
SPME fibre assembly polydimethylsiloxane/divinylbenzene (df 65 µm, Fused Silica/SS)
90
(PDMS/DVB), potassium persulphate, n-alkanes (C8–C22), 4-octanol, 500 µm fine test sieve, and
91
9.00 cm Whatman filter paper (No. 4) were all purchased from Sigma-Aldrich (Castle Hill, NSW,
92
Australia). Methanol, sodium carbonate anhydrous, sodium nitrate, and sodium hydroxide pellets
93
were from Chem-Supply (Gillman, SA, Australia). Hydrochloric acid 32% was obtained from
94
Science Supply (Mitcham, VIC, Australia). Headspace screwtop clear vials (20 ml) and magnetic
95
PTFE/sil hdsp cap were purchased from Agilent Technologies (Singapore). All chemicals were of
96
analytical reagent or HPLC grade.
TE D
EP
AC C
97
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85
4
ACCEPTED MANUSCRIPT
98
2.3. Analysis of phenolic contents and antioxidant activity
99
2.3.1. Sample preparation About 20 g of each collected sorghum grain sample was freeze-dried at -20°C for 6 days. Then 5 g
101
of each dried sorghum grain sample was separately ground and sieved (100% through a 500 µm
102
sieve) to obtain sorghum powder. All the results in this study were reported on a dry matter basis.
RI PT
100
103 2.3.2. Extraction of phenolic contents
105
The extraction of phenolic compounds was performed using the method described by Wu et al.
106
(2013) with slight modifications. Briefly, dried sorghum powder (1 g) was extracted with 45 ml of
107
80% methanol solution (v/v) for 1 h at 50 °C, and then centrifuged at 3,220 g and 4°C for 10 mins.
108
After centrifugation, the supernatant was collected, and the residue was re-extracted one more time
109
under the same conditions. The supernatants were pooled and evaporated at 50 °C to dryness using
110
a rotary evaporator, then reconstituted in 10 ml methanol to give the phenolic extract solution. The
111
extract solution was then stored at -20 °C in dark until analysis.
M AN U
TE D
112
SC
104
2.3.3. Determination of total phenolic content (TPC)
114
TPC was measured using the Folin-Ciocalteu method described by de la Rosa, Alvarez-Parrilla, and
115
Shahidi (2010). An aliquot of 60 µl of the extract solution and 60 µl methanol were mixed with 750
116
µl of 10% Folin-Ciocalteu’s reagent solution (v/v) and incubated for 2 mins at room temperature.
117
Then, 600 µl of 7.5% (w/v) sodium carbonate solution was added, and the mixture was incubated at
118
50 °C for 20 min in the dark and then cooled to room temperature. The absorbance was measured
119
by a UV-Vis spectrophotometer (M501 Single Beam Scanning, Camspec, Leeds, UK) at 760 nm.
120
Gallic acid was used as the standard and methanol as blank, and the results were expressed as mg
121
gallic acid equivalents (GAE)/g dry basis (db).
AC C
EP
113
122
5
ACCEPTED MANUSCRIPT
2.3.4. Determination of total flavonoid content (TFC)
124
TFC was measured by the aluminium chloride colourimetric method according to de la Rosa,
125
Alvarez-Parrilla, and Shahidi (2010) with a slight modification. An aliquot of 150 µl of the extract
126
solution was mixed with 660 µl of water and 45 µl of 5% NaNO2 solution (w/v) and incubated for 5
127
min at room temperature. Then 45 µl of 10% AlCl3 solution (w/v) was added to the mixture and
128
incubated for 3 min at room temperature. Next, 600 µl of 0.5 M NaOH solution was added, and the
129
mixture was incubated for 30 min at room temperature in the dark. The absorbance was measured
130
by the M501 UV-Vis spectrophotometer at 415 nm. (+)-catechin hydrate was used as the standard
131
and methanol as the blank, and the results were expressed as mg catechin equivalents (CAE)/g db.
SC
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123
M AN U
132 2.3.5. Determination of condensed tannin content (CTC)
134
CTC was determined by the vanillin-HCl assay method described by Wu et al. (2016) with a slight
135
modification. The vanillin-HCl reagent was prepared daily, by mixing an equal volume of 2%
136
vanillin in methanol solution (w/v) with acidified methanol solution (8% HCl, v/v). An aliquot of
137
800 µl of the extract was mixed with 0.700 µl of vanillin-HCl reagent. The resulting solution was
138
mixed and incubated for 20 min at room temperature, and the absorbance was measured at 500 nm
139
by the M501 UV-Vis spectrophotometer. Catechin was used as the standard and methanol as the
140
blank, and the results were expressed as mg catechin equivalents (CAE)/g db.
EP
AC C
141
TE D
133
142
2.3.6. Determination of DPPH radical scavenging activity (DPPH)
143
Radical DPPH scavenging activity was measured according to the method of Wu, Johnson,
144
Bornman, Bennett, and Fang (2017) with a slight modification. About 24 mg of DPPH was added
145
to 100 ml methanol to prepare the stock solution, and then stored at -20°C in the dark. About 8 ml
146
stock solution was diluted with 40 ml methanol to prepare the working DPPH solution with an
147
absorbance of 1.1 ± 0.02 units at 515 nm. The sorghum extract was diluted ten times with methanol,
148
then 0.150 ml of the diluted sorghum extract was mixed with 1.350 ml freshly prepared the DPPH 6
ACCEPTED MANUSCRIPT
149
working solution. The resulting solution was incubated for 8 h at room temperature in the dark, and
150
the absorbance was measured at 515 nm. Trolox was used as the standard and methanol as the blank,
151
and results were expressed as mg Trolox equivalents (TE)/g db.
152 2.3.7. Determination of ABTS radical scavenging activity (ABTS)
154
The ABTS assay was conducted following the method of Wu et al. (2017). Equal amounts of 7.4
155
mM ABTS•+ solution and 2.6 mM potassium persulphate solution were mixed and reacted for 12 h
156
at room temperature in the dark to give the stock solution. The ABTS•+ working solution was
157
freshly prepared, by diluting 0.8 ml of the stock solution with 50 ml methanol to get an absorbance
158
of 1.1 ± 0.02 units at 734 nm, measured by the M501 UV-Vis spectrophotometer. The sorghum
159
extract was diluted 10 times with methanol, then 0.150 ml of the diluted sorghum extract was
160
reacted with 1.350 ml ABTS•+ working solution for 2 h at room temperature in the dark, and then
161
the absorbance was measured at 734 nm. Trolox was used as the standard and methanol as the blank,
162
and results were expressed as mg Trolox equivalents (TE)/g db.
163
TE D
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153
2.4. Analysis of volatile compounds
165
2.4.1. Moisture content
166
Moisture content was measured by drying 3 g of each sorghum sample in an oven at 105 °C until a
167
constant weight was reached. Moisture content was used to quantify the volatile compounds on the
168
dry basis.
AC C
169
EP
164
170
2.4.2. Sample preparation and headspace solid-phase microextraction (HS-SPME) procedures
171
The extraction of volatile compounds was carried out by HS-SPME method, using a 65 µm
172
PDMS/DVB fibre equipped with an SPME PAL 3 multi-purpose automated sampler (Agilent,
173
USA). Before analysis, the fibre was pre-conditioned at 250 °C for 30 min in the GC injection port
174
in accordance with manufacturer’s specifications. 7
ACCEPTED MANUSCRIPT
176
The sorghum volatile compounds were extracted according to the method of Zanan et al. (2016).
177
Raw, soaked, steamed and roasted sorghum grains were ground into powder with liquid nitrogen.
178
Two gram of sorghum powder was weighted and immediately transferred into a 20-ml headspace
179
vial, added with 20 µL of internal standard 4-octanol (0.01 g/100 ml) and then tightly sealed with a
180
magnetic PTFE/sil cap. The vial was shaken incubated at 80 °C for 20 min for equilibrium, and the
181
SPME fibre was then exposed to the headspace at 80 °C for 35 min for adsorption.
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To analyse sorghum tea volatile compounds, both roasted sorghum grain and its ground powder
184
were infused by boiling water as described by Lv, Zhong, Lin, Wang, Tan, and Guo (2012). Briefly,
185
about 2 g of whole roasted grain or ground powder was weighted and immediately transferred into a
186
20-ml headspace vial, added with 20 µL of internal standard 4-octanol (0.01 g/100 ml) and infused
187
with boiling water (10 ml, 100 °C) respectively. The vial was tightly sealed immediately with a
188
magnetic PTFE/silicon cap, agitated for 10 min to reach equilibrium. Then, the SPME fibre was
189
exposed to the headspace and kept at 60 °C for 60 min.
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2.4.3. Gas chromatography-mass spectrometry (GC-MS) analysis
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The GC-MS analysis was carried out following the method described by Lv, Zhong, Lin, Wang,
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Tan, and Guo (2012) with slight modifications. A gas chromatograph (Agilent Technologies 6850
194
series II gas chromatograph, USA) was coupled to a mass spectrometer (Agilent Technologies 5973
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mass selective detector, USA) and a J&W DB-5ms capillary column (30 m x 250µm x 0.25µm)
196
was used.
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After extraction, the SPME fibre was introduced to the GC-MS analysis. SPME fibre desorption
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was conducted at GC injection port in splitless mode, with an injection temperature 250 °C. Helium
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(percentage purity > 99.999%) was used as the carrier gas at a constant flow velocity of 1 ml/min. 8
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The oven temperature program was set as follows: an initial temperature of 50 °C and held for 5
202
min, then increased to 125 °C at a rate of 3 °C/ min and held for 3 min; and then increased to
203
180 °C at a rate of 2 °C/min and held for 3 min; and last increased to the final temperature 230 °C at
204
a rate of 15 °C/min and held for 3 min. The mass spectrometer was operated in ionisation mode at
205
70 eV, with a mass scan range of 35-400 AMU; the interface, ion source and quadrupole
206
temperature were 280 °C, 230 °C and 150 °C, respectively. The solvent delay time was 3.0 min.
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2.4.4. Compound identification and quantification
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The GC-MS data were analysed using Agilent G1701EA MSD ChemStation software (Version
210
1.4.20.0). The linear retention index (RI) of each compound was calculated using a series of n-
211
alkanes (C8–C22). Each compound was identified by comparing the mass spectra and linear
212
retention indices (RI), using the NIST reference database (NIST 11.0) and NIST Chemistry
213
Webbook (NIST, 2017), where available. The concentration of each compound was reported semi-
214
quantitatively as internal standard (I.S.) equivalents to compare relative concentration changes
215
among the studied samples only. The spectrums were scanned in the total ion chromatogram (TIC)
216
mode. Each identified volatile compound was quantified by comparing its ion peak area with
217
internal standard 4-octanol. The concentration was calculated on a dry basis by considering the
218
moisture content and using the following equation according to Xiao, Lee, Zhang, Ebeler,
219
Wickramasinghe, Seiber, et al. (2014) as follows:
220
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µ
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ℎ 1 µ = × . . . ×
. . (1 − )
221 222
2.5. Statistical analysis
223
The sorghum tea processing was repeated three times, and the analysis of phenolic content and
224
antioxidant activity was done in triplicates on each repeated sample (a total of 9 replicates). The
225
analysis of moisture contents and volatile compounds was done on each repeated sample (a total of 9
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3 replicates). The significant difference of means between each processing step was determined
227
using one-way ANOVA with Turkey grouping at 95% confidence level (Minitab Express, version
228
1.2.0, Sydney, Australia). Due to the large amount of background noise and the impurity peak in the
229
chromatograms of grain infusion sample, an additional one-way ANOVA test was performed for
230
the data of total aldehyde and total pyrazine with the grain infusion sample data removed.
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231 3. Results and Discussion
233
3.1. Phenolic contents
234
It was reported that soaking could alter the physical structure and functionality of grains (Ebuehi &
235
Oyewole, 2008). Prolonged soaking increases the water content and leads to rupture of grain cells,
236
releasing the cellular contents, which further accelerates the leaching of water-soluble compounds
237
(Ituen, Mittal, & Adeoti, 1986; Wu et al., 2013). However, in the present study, the soaking process
238
increased the TFC in sorghum while TPC and CTC were not affected (Table 1). A possible
239
explanation for this might be that the white sorghum variety (Liberty) used in this study had
240
relatively lower phenolic contents as compared to other sorghum genotypes such as red and brown
241
sorghums (Wu et al., 2017), and most of the phenolic compounds in sorghum are in the bound form
242
(Dykes, Rooney, Waniska, & Rooney, 2005; Wu et al., 2017). Minor leaching of phenolic
243
compounds might have occurred during the soaking process, but the loss of TPC and CTC might be
244
negligible to cause statistical significant differences. The slight increase of TFC might be due to the
245
release of some bound phenolic compounds when the water penetrated into cell walls, as the above
246
method (Section 2.3.2) were mainly able to extract free phenolic compounds (Wu et al., 2013).
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Steaming can severely damage the cellular structure of grains, which may cause the release of some
249
bound phenolic compounds (Randhir, Kwon, & Shetty, 2008; Wu et al., 2013). In addition, the heat
250
effect during the steaming process can also lead to the degradation of some phenolic compounds
251
such as vanillic and p-coumaric acids (Wu et al., 2013). Interestingly, our results showed that the 10
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steaming process increased (P ≤ 0.05) TFC in sorghum but had no significant (P > 0.05) effect on
253
TPC and CTC (Table 1). The reason could be similar to the above soaking process that there was a
254
relatively lower phenolic content in this sorghum variety. Some phenolic compounds might be
255
damaged during the process, while some bound phenolic compounds might be released, so the
256
overall phenolic compounds remained unchanged. Wu et al. (2013) also reported elevated free
257
ferulic acid and bound p-coumaric acid levels in the steamed red sorghum grain.
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The final sorghum grain tea product was obtained after the roasting process, which slightly
260
increased the TPC and CTC, while TFC (P > 0.05) was significantly enhanced compared to the
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steamed samples (Table 1). In comparison with the initial raw sorghum, the TPC, TFC and CTC in
262
roasted sorghum were all significantly (P ≤ 0.05) higher. The result is consistent with that of red
263
sorghum grain tea processing as reported by Wu et al. (2013). Two mechanisms have been
264
proposed: (1) roasting can cause damage to cell wall and break down cellular constituents, therefore,
265
releases some bound phenolic compounds (Randhir, Kwon, & Shetty, 2008); (2) at high
266
temperature, some high molecular weight or polymeric phenolic compounds such as condensed
267
tannins are break down into simple and extractable molecules (Cheng, Su, Moore, Zhou, Luther,
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Yin, et al., 2006).
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3.2. Antioxidant activity
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It is widely recognised that the grain phenolic compounds are the main contributors to antioxidant
272
activity (de Morais Cardoso, Pinheiro, Martino, & Pinheiro-Sant'Ana, 2017; Dykes & Rooney,
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2006; Stefoska-Needham, Beck, Johnson, & Tapsell, 2015). The ABTS and DPPH radical
274
scavenging activity of the extracts from raw and processed sorghum grains are shown in Table 1,
275
which suggests that the soaking, steaming and roasting process did not affect (P > 0.05) the ABTS
276
and DPPH radical scavenging activity. Although the above results indicated that the TPC, TFC and
277
CTC in the roasted sorghum were significantly (P ≤ 0.05) higher than the raw sorghum, the
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antioxidant activity remained unchanged, implying that the relatively low phenolic compounds in
279
this white sorghum variety might have less impact on its antioxidant activity compared to coloured
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sorghum, other compounds might also contribute to the antioxidant activity. It was reported that
281
vitamins, peptides and proteins also have strong antioxidant activities (Elias, Kellerby, & Decker,
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2008; Gliszczyńska-Świgło, 2006), and their contributions to the antioxidant activity of the present
283
sorghum variety need further investigation.
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284 3.3. Sorghum volatile profile
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The volatile compounds of raw and processed sorghum grain and sorghum grain tea infusions are
287
summarised in Table 2, with representative GC-MS chromatogram of raw sorghum and final
288
sorghum tea product (roasted sorghum) presented in Fig. 2. This represents the very first research
289
on sorghum tea volatile profiling. Since the volatile profiles were measured in different forms of
290
products, including solid raw sorghum, intermedium processed products and liquid tea product, it is
291
unlikely to construct chemical standard calibration curves to accurately simulate each product
292
condition. Semi-quantification was used in this study for the profiling of sorghum derived volatiles,
293
which could effectively achieve the purpose of the current study to investigate the influences of
294
processing technology on sorghum volatile profile. A total of 63 volatile compounds were detected
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from the sorghum samples. Amongst those compounds, 45 were identified based on mass spectra
296
(MS) and retention index (RI) from the in-house NIST library 11.0 and NIST webbook (NIST,
297
2017), while 18 were unidentified. The identified volatile compounds were classified into eight
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chemical groups including 5 alcohols, 13 alkanes, 2 aldehydes, 2 carboxylic acids, 15 esters, 4
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ketones, 3 pyrazines and 1 phenylenediamine. The unidentified compounds had distinct but weak
300
signals (Fig. 2) and unidentifiable by the NIST (11.0) MS database. However, the MS information
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indicated that these compounds were most likely to be 10-12 carbon alkanes or its derivatives.
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3.4. Raw sorghum volatile compound profile
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3.4.1. Esters
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Esters were the dominant volatile compounds in raw sorghum, and the total ester content was 33.83
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± 1.35 µg/g db (Table 2). Amongst the 15 detected esters, methyl esters were the dominant ester in
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tested sorghum. These esters contributes to fruity, green, sweet, and floral aroma and flavour of
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food and beverages (Acree & Arn, 2017; TGSC, 2017).
309
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3.4.2. Alkanes
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Alkanes were the second most abundant volatile compounds in the tested sorghum (Table 2).
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Thirteen alkanes were detected in raw sorghum, and the total alkane content was 15.28 ± 0.61 µg/g
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db. In general, alkanes have waxy and gasoline-like odour (Acree & Arn, 2017; TGSC, 2017).
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Although the alkane content in sorghum was high, it might not make a significant contribution to
315
aroma due to their low odour intensity and high odour threshold values (He, Guo, Yang, Xie, Ju, &
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Guo, 2016; Zanan et al., 2016). Alkanes, aldehydes and alkenes are associated with lipid oxidation
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products, and high concentrations of these compounds may cause off-flavour in food
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products(Bryant & McClung, 2011).
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3.4.3. Alcohols
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Alcohols were the third most abundant volatile compounds (13.31 ± 0.61 µg/g db) in sorghum
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(Table 2). Five alcohols including 1-hexanol (1), 1-octanol (13), 1,6-octadien-3-ol, 3,7-dimethyl-
323
(17), phenylethyl alcohol (19) and 1-nonanol (25) were detected, with a noticeable high
324
concentration (8.95 ± 0.44 µg/g db) of 1-hexanol. These alcohols are associated with intense sweet,
325
floral, waxy, green, citrus and fruity odour descriptions (Acree & Arn, 2017; TGSC, 2017).
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3.4.4. Other compounds
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Other compounds including aldehydes, carboxylic acids and ketones were detected in small
329
quantities in sorghum (Table 2). Aldehydes of nonanal (18) and 2,4-decadienal (47) were
330
determined, in which nonanal (18) has waxy, aldehydic and citrus odour, and 2,4-decadienal (47)
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has fatty, oily and chicken skin-like odour (TGSC, 2017). The carboxylic acid of hexanoic acid (5)
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is responsible for fatty, sweat and cheese odour, and the ketones of 2(3H)-furanone, 5-ethyldihydro-
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(10) and 2(3H)-furanone, dihydro-5-pentyl- (50) have pleasant sweet and creamy-like aroma
334
(TGSC, 2017).
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3.5. Effect of soaking processing on the sorghum volatile compounds
337
The purpose of soaking was to increase the grain moisture content, swell and soften the physical
338
structure, thus to improve the subsequent thermal treatment efficiency. Surprisingly, soaking had a
339
great impact (P ≤ 0.05) on the volatile compounds (Table 2). The TVC (161.72 ± 26.42 µg/g db)
340
was increased about two folds compared to the raw sorghum, which was mainly contributed by the
341
ester compounds as the total ester content (122.16 ± 27.25 µg/g db) increased approximately four
342
folds compared to the raw grain. It was suggested that soaking can lead to a high cell stress and
343
consequent rupture and thus result in more efficient extraction of esters (Ituen, Mittal, & Adeoti,
344
1986; Wu, et al., 2013). The enzymatic esterification reactions of short chain acids and alcohols
345
might have also contributed to the increased esters during the soaking process because sufficient
346
water content could activate the lipase activity to produce esters (Gubicza, Kabiri-Badr, Keoves, &
347
Belafi-Bako, 2000; Wehtje, Kaur, Adlercreutz, Chand, & Mattiasson, 1997).
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Alkanes were the second most abundant volatile compounds in soaked sorghum grains. The total
350
alkane content was 17.74 ± 1.69 µg/g db, which was higher than the raw sorghum (P ≤ 0.05).
351
Decane, 4-methyl- (9), decane, 3,7-dimethyl- (11), undecane, 2,6-dimethyl- (30), tridecane (46) and
352
pentadecane (59) were the most abundant alkanes in soaked sorghum. A significant (P ≤ 0.05) 14
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increase of aldehydes, especially nonanal (18), was also observed in the soaked sorghum compared
354
to the raw seed. Alkanes are a type of plant lipid and water-insoluble, which are mainly located in
355
the germ of grains and can be difficult to extract (Cheek & Jebb, 2001; Waniska & Rooney, 2000).
356
The results indicated that soaking sorghum for 17 h could increase the extraction efficiency of
357
alkanes as well as the aldehydes.
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The alcohol content (5.14 ± 0.15 µg/g db) was significantly (P ≤ 0.05) reduced in the soaked
360
sorghum compared to the raw (Table 2). In addition, carboxylic acids eg. hexanoic acid (5), which
361
were relatively high in raw sorghum but were not detected in the soaked sorghum. Similarly, ketone
362
levels were also greatly lowered, and 2(3H)-furanone, 5-ethyldihydro- (10) was not detected in the
363
soaked sorghum. The reason could be that alcohols, carboxylic acids and ketones especially small
364
molecular weight (MW) molecules such as 1-hexanol (1) and hexanoic acid (5) are water-soluble,
365
and may have been leached out during the soaking process (Lucas, Le Ray, & Mariette, 2007). The
366
reduction of alcohol content also partly supported the postulation that they might have been
367
involved in esterification reactions.
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3.6. Effect of steaming processing on the sorghum volatile compounds
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The steaming process dramatically reduced the TVC (Table 2) in contrast to the soaking process.
371
The TVC in steamed sorghum was 52.71 ± 4.60 µg/g db which was about three folds lower than in
372
the steamed grain, with this reduction being mainly due to the loss of ester content. After the
373
steaming process, some esters found in soaked grain were not detected, and the most abundant ester
374
compounds (i.e. peak 62, 63 and 64) that were previously found in raw and soaked sorghum were
375
barely detectable. The alcohol content was also reduced by the streaming process, and phenylethyl
376
alcohol (19) found in the raw and soaked grains was not detected in the steamed sorghum. A
377
possible explanation for this might be the decomposition of esters and leaching of alcohols. Esters
378
are only very slightly soluble in water and might not be affected by leaching, but they could have
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379
evaporated from the sample during the steaming process or have been hydrolysed into fatty acids
380
and alcohols (Rodrigues & Vale, 2009).
381 The alkane and aldehyde contents of the grains were further increased (P ≤ 0.05) upon steaming.
383
Alkane was the most abundant volatile compound in the steamed sorghum, with a total
384
concentration of 23.03 ± 2.41 µg/g db, and aldehyde had a total concentration of 3.50 ± 0.09 µg/g
385
db. The steaming process can damage the cellular structure of the grains, and thus might have led to
386
the increased extractability (Randhir, Kwon, & Shetty, 2008; Wu et al., 2013). This may also have
387
facilitated lipid oxidation thus more production of alkane and aldehyde (Bryant and McClung,
388
2011). Another reason of high abundance of alkane in steamed sorghum might be that these
389
alkanes were mostly 10-12 carbon and are relatively stable during wet heat processing.
390
Nevertheless, the carboxylic acid and ketone contents were not affected by the steaming process,
391
indicating they were stable under the steaming conditions used.
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3.7. Effect of roasting processing on the sorghum volatile compounds
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The roasted sorghum is the final sorghum tea product ready for infusion. Although the roasting
395
process had no significant (P > 0.05) effect on the TVC (47.26 ± 4.42 µg/g db), the profile of
396
volatile compounds was changed (Table 2). For example, pyrazines were only detected after the
397
roasting process. The total pyrazine content in the roasted sorghum was 3.06 ± 0.29 µg/g db, which
398
included pyrazine 2,5-dimethyl- (2), pyrazine, 2-ethyl-3, 5-dimethyl- (14) and pyrazine,
399
tetramethyl- (15). These pyrazines generally have roasted and nutty odour attributes (TGSC, 2017).
400
It has been suggested that pyrazines can be biosynthesized by microorganisms such as Bacillus
401
cereus or by heating processing (Adams & De Kimpe, 2007; Guo, et al., 2017). During the heating
402
process, pyrazines are formed by Maillard reactions of amino acids with reducing sugars and
403
Strecker degradations (Alasalvar, Shahidi, & Cadwallader, 2003). In addition, 1,2-benzenediamine,
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404
4-methyl- (7) was also only found in the roasted sorghum, and this compound is likely to be
405
produced via a similar mechanism as pyrazine and possibly contributed to the roasted aroma.
406 Other volatile compounds such as alcohol, alkane, aldehyde, ester, carboxylic acid and ketone
408
contents remained relatively stable during the roasting process. However, compared to the raw
409
sorghum, the aldehyde and alkane contents in the roasted sorghum were slightly higher, while the
410
alcohol, ester, carboxylic acid and ketone contents were significantly lower, and many of them were
411
not detected in the roasted sorghum (Table 2). Processing has a great impact on the volatile profile
412
of sorghum grain and the mechanism needs further investigation.
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415
The volatile compounds in the roasted sorghum whole grain infusion (GI) and grain powder
416
infusion (PI) were compared (Table 2). The TVC in PI was significantly higher (P>0.05) than in GI,
417
and more individual volatile compounds were detected in the PI tea. The results suggest that the
418
extraction of volatile compounds in the powder form infusion tea was more effective than the whole
419
grain form infusion tea. Therefore, the powder form of roasted sorghum grain could be more
420
suitable for sorghum tea making. In addition, this roasted sorghum grain powder could be further
421
processed into consumer-friendly forms such as tea bags. However, it should be noted that no
422
significant (P > 0.05) difference in TVC was observed between these two type teas (Table 2). This
423
was due to the significant data variation, which was caused by the interference of background noise
424
and impurity peaks in the chromatograms of the infusion samples, and these impurity peaks were
425
identified as plastic materials and might come from the headspace vial and cap materials under the
426
prolonged infusion of boiling water. A methodology development, such as by varying extraction
427
temperature, time and fibre type, is required to optimise the characterisation of infusion volatiles in
428
the future study.
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Regardless of the form of grain used for infusion, a significant increase of the pyrazine and
431
phenylenediamine contents was observed upon infusion as compared to the roasted sorghum grain.
432
The increase of the pyrazine contents might be due to the fact that pyrazine is water soluble, and
433
infusion with boiling water might cause swelling of grains and thus releasing pyrazine from the
434
matrix (Ituen, Mittal, & Adeoti, 1986; Wu et al., 2013). Another possible explanation is that heating
435
by boiling water during the infusion could lead to Maillard reaction and thus generate more
436
pyrazines (Morini & Maga, 1995).
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437 4. Conclusion
439
In this study, a sorghum grain tea was developed using a white sorghum variety (Liberty) through
440
three consecutive processes of soaking, steaming and roasting. Compared to the raw sorghum grain,
441
the phenolic contents (TPC, TFC, CTC) in the roasted sorghum were significantly higher, although
442
antioxidant activity (DPPH, ABTS) was not affected, suggesting other compounds might have
443
contributed to the antioxidant activity of the grains. In terms of the volatile compounds, a total of 63
444
volatile compounds including 5 alcohols, 13 alkanes, 2 aldehydes, 2 carboxylic acids, 15 esters, 4
445
ketones, 3 pyrazines and 1 phenylenediamine were detected in the sorghum samples. The soaking
446
process dramatically increased the ester content as well as alkane, aldehyde, while reduced the
447
alcohol, carboxylic acids and ketone contents. Steaming, however, significantly reduced the ester
448
and alcohol contents, while increased the alkane and aldehyde contents. The roasting process
449
resulted in the formation of pyrazine and phenylenediamine compounds. In addition, powder form
450
infusion was more effective in extracting volatile compounds than whole grain form infusion,
451
therefore the powder form may be more suitable for sorghum tea making. According to the detected
452
volatile compounds, the white sorghum tea may have an overall floral, sweet, waxy, and nutty
453
aroma. Future studies are required to compare the effect of processing on different sorghum
454
varieties (e.g. red and black sorghums which have higher phenolic contents), as well as sensory
455
evaluation of the infused sorghum grain tea products.
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456 457
5. Conflict of interest
458
None.
459 6. References
461
Acree, T., & Arn, H. (2017), Flavornet, Kovats RI,
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Oct 2017.
Adams, A., & De Kimpe, N. (2007). Formation of pyrazines and 2-acetyl-1-pyrroline by Bacillus cereus.
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Alasalvar, C., Shahidi, F., & Cadwallader, K. R. (2003). Comparison of natural and roasted Turkish tombul
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hazelnut (Corylus avellana L.) volatiles and flavor by DHA/GC/MS and descriptive sensory analysis.
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Phytochemistry, 65(9), 1199-1221.
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Chemistry, 59(1), 152-162.
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de Morais Cardoso, L., Pinheiro, S. S., Martino, H. S. D., & Pinheiro-Sant'Ana, H. M. (2017). Sorghum
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Dykes, L., & Rooney, L. W. (2006). Sorghum and millet phenols and antioxidants. Journal of Cereal Science, 44(3), 236-251. Dykes, L., Rooney, L. W., Waniska, R. D., & Rooney, W. L. (2005). Phenolic compounds and antioxidant
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Ebuehi, O. A. T., & Oyewole, A. C. (2008). Effect of cooking and soaking on physical characteristics,
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Elias, R. J., Kellerby, S. S., & Decker, E. A. (2008). Antioxidant activity of proteins and peptides. Critical reviews in food science and nutrition, 48(5), 430-441.
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EP
499
Guo, H., Yang, X., Zhou, H., Luo, X., Qin, P., Li, J., & Ren, G. (2017). Comparison of Nutritional
503
Composition, Aroma Compounds, and Biological Activities of Two Kinds of Tartary Buckwheat
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502
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He, C., Guo, X., Yang, Y., Xie, Y., Ju, F., & Guo, W. (2016). Characterization of the aromatic profile in
506
“zijuan” and “pu-erh” green teas by headspace solid-phase microextraction coupled with GC-O and
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Ituen, E., Mittal, J., & Adeoti, J. (1986). Water absorption in cereal grains and its effect on their rupture stress. Journal of Food Process Engineering, 8(3), 147-158. Kraujalytė, V., Pelvan, E., & Alasalvar, C. (2016). Volatile compounds and sensory characteristics of various instant teas produced from black tea. Food Chemistry, 194, 864-872.
20
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Lucas, T., Le Ray, D., & Mariette, F. (2007). Kinetics of water absorption and solute leaching during soaking of breakfast cereals. Journal of food engineering, 80(2), 377-384.
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Lv, H.-P., Zhong, Q.-S., Lin, Z., Wang, L., Tan, J.-F., & Guo, L. (2012). Aroma characterisation of Pu-erh
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516
Chemistry, 130(4), 1074-1081.
520 521 522
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Randhir, R., Kwon, Y.-I., & Shetty, K. (2008). Effect of thermal processing on phenolics, antioxidant
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TE D
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okadaite's esters. Toxicon, 53(7), 806-809.
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529
cereal whole grain with the potential to assist in the prevention of chronic disease. Food Reviews
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532 533 534 535 536
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AC C
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EP
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Accessed 17 Oct 2017.
Waniska, R. D., & Rooney, L. W. (2000). Structure and chemistry of the sorghum caryopsis. Sorghum: Origin, history, technology, and production, 2, 649-679. Wehtje, E., Kaur, J., Adlercreutz, P., Chand, S., & Mattiasson, B. (1997). Water activity control in enzymatic esterification processes. Enzyme and microbial technology, 21(7), 502-510.
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538
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541
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543 544
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547
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549
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548
RI PT
545
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551
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552
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Figure captions
554
Fig. 1. Flow diagram of sorghum grain tea processing, sampling and analysis.
555
Fig. 2. GC-MS chromatograms showing the volatile compound profile of raw (a) and roasted (b)
556
sorghum. Peak numbers are identified in Table 2; peak No. 6 is the internal standard (I.S.) 4-octanol.
AC C
EP
TE D
M AN U
SC
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23
SC
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
Fig. 1 Flow diagram of sorghum grain tea processing, sampling and analysis.
AC C
558
M AN U
557
24
EP
559
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 2. GC-MS chromatograms showing the volatile compound profile of raw (a) and roasted (b)
561
sorghum. Peak numbers are identified in Table 2; peak No. 6 is the internal standard (I.S.) 4-octanol.
AC C
560
25
ACCEPTED MANUSCRIPT
562
Table 1: Effect of processing on TPC, TFC, CTC, and ABTS and DPPH radical scavenging activity
563
of sorghum grain. Analysis
Processing Soaked
Steamed
Roasted
TPC
0.34 ± 0.02b
0.33 ± 0.02b
0.36 ± 0.02ab
0.41 ± 0.03a
TFC
0.71 ± 0.02d
0.78 ± 0.02c
0.89 ± 0.03b
0.94 ± 0.03a
CTC
3.26 ± 0.18b
3.13 ± 0.20b
3.44 ± 0.18ab
3.89 ± 0.33a
ABTS
2.01 ± 0.15a
1.95 ± 0.09a
1.94 ± 0.20a
2.14 ± 0.09a
DPPH
1.42 ± 0.07a
1.39 ± 0.10a
1.37 ± 0.09a
1.49 ± 0.15a
SC
RI PT
Raw
TPC = total phenolic content (mg GAE/g db).
565
TFC = total flavonoid content (mg CAE/g db).
566
CTC = condensed tannin content (mg CAE/g db).
567
ABTS and DPPH = ABTS and DPPH radical scavenging activity (mg TE/g db).
568
a, b, c
M AN U
564
AC C
EP
TE D
= values with different superscripts in the same row are significantly different (P ≤ 0.05).
26
ACCEPTED MANUSCRIPT
569
Table 2: Effect of processing and infusion on sorghum grain tea volatile compounds (µg/g sample
570
db). Pea
Compound Name
RI*
k
Processing Raw
Soaked
Steamed
Roasted
No. 1
1-Hexanol
867
8.95±0.44
a
2.70±0.17
b
0.82±0.10
c
ND
Powder
Grain
infusion
infusion
ND
ND
ID**
Aroma***
RI,
Pungent, ethereal, fruity
MS 1-Octanol
107
0.60±0.03a
0.48±0.07a
0.19±0.02b
0.18±0.01b
0.19±0.02b
0 17
19
1,6-Octadien-3-ol, 3,7-
109
dimethyl-
9
Phenylethyl Alcohol
110
2.17±0.14a
1.52±0.10ab
2.21±0.17a
1.75±0.19ab
1.8±0.21a
1.08±0.54b
117
0.73±0.04a
0.19±0.02b
ND
ND
ND
ND
0.86±0.07a
0.25±0.02b
13.31±0.61
102
0.14±0.02bc
5.14±0.15
b
3.36±0.09
0.97±0.04b
1.40±0.12a
c
1.77±0.20a
2 11
Decane, 3,7-dimethyl-
105
Dodecane, 2,6,10-
106
trimethyl-
0
23
Undecane, 2-methyl-
116
28
Dodecane
120
29
Undecane, 2,4-
0.12±0.02c
2.05±0.21
d
dimethyl-
0
30
Undecane, 2,6-
121
dimethyl-
3
Undecane, 4,8-
122
dimethyl-
2
Dodecane, 4,6-
125
dimethyl-
2
46
Tridecane
129
RI,
2.23±0.20
cd
1.68±0.24a
1.47±0.73
Aldehydic, waxy, citrus, tart,
MS
sweet
RI,
--
d
ND
MS
2.82±0.40a
2.84±0.41a
0.32±0.17c
MS
--
0.72±0.03b
1.03±0.09a
1.29±0.14a
1.14±0.13a
1.16±0.18a
0.14±0.07c
MS
--
0.52±0.05c
0.55±0.06bc
0.81±0.13a
0.71±0.10ab
0.66±0.10abc
ND
RI,
--
1.09±0.07b
1.16±0.15ab
1.51±0.17a
1.49±0.17a
1.30±0.16ab
0.37±0.17c
MS
Alkane
--
TE D
0.36±0.02c
MS
0.39±0.07bc
0.53±0.06a
0.47±0.05ab
0.38±0.03c
ND
RI,
1.34±0.25ab
1.83±0.32a
1.64±0.28a
1.35±0.10ab
0.13±0.05c
MS
--
0.77±0.10ab
1.00±0.14a
0.95±0.13a
0.75±0.11ab
ND
MS
--
0.90±0.13ab
1.08±0.16ab
1.04±0.14a
0.72±0.09c
ND
MS
--
4.79±0.11b
4.79±0.22b
7.97±0.72a
6.79±0.19a
5.57±0.70b
0.40±0.20c
MS
Gasoline-like to odourless
0.76±0.07ab
0.89±0.12ab
0.97±0.12a
0.76±0.08bc
0.54±0.04c
0.24±0.09d
MS
Gasoline-like, mild waxy
0.76±0.08b
0.84±0.24a
0.43±0.05c
0.37±0.09cd
0.20±0.10d
ND
MS
Waxy
0.59±0.05c
1.16±0.13a
0.75±0.00b
0.40±0.04d
0.24±0.04d
ND
MS
Waxy
15.28±0.61c
17.74±1.69ab
23.03±2.41
20.16±1.89a
17.39±2.29bc
1.60±0.74d
a
b
RI,
Waxy, aldehydic, citrus
1.27±0.11b
EP
0.68±0.08b
0.85±0.07bc
AC C
36
0.15±0.10bc
3.08±0.30a
0
31
1.59±0.23a
0.15±0.01bc
2.51±0.22a
4
121
Sweet, floral, fresh, bready
1.92±0.16b
4 12
RI,
M AN U
Decane, 4-methyl-
Citrus, orange, floral, waxy, rose
MS
a
9
Waxy, green, citrus, floral
MS
1 Total alcohol
RI,
SC
1-Nonanol
RI, MS
8 25
0.24±0.13b
RI PT
13
MS
9
54
Tetradecane
139
9
58
Heneicosane
148
5
59
Pentadecane
149 9
Total alkane
18
Nonanal
110
47
2,4-Decadienal
131
0.54±0.03b
1.67±0.11ab
2.62±0.13ab
1.39±0.14ab
2.76±0.10ab
3.66±2.26a
0.04±0.01a
0.13±0.01a
0.87±0.05a
0.72±0.14a
2.12±0.17a
5.98±6.99a
0.58±0.03a
1.80±0.11aC
3.50±0.18aB
2.12±0.28aC
4.88±0.07aA
9.64±9.25a
3
MS
MS
5 Total aldehyde
RI,
27
Fatty, oily, chicken skin-like
ACCEPTED MANUSCRIPT
D
5
Hexanoic acid
979
2.09±0.52
ND
ND
ND
ND
ND
RI,
Fatty, sweat, cheese
MS 37
0.78±0.04c
0.89±0.04bc
1.24±0.11a
0.99±0.10ab
0.96±0.07bc
0.18±0.12d
2.87±0.51a
0.89±0.04b
1.24±0.11b
0.99±0.10b
0.96±0.07b
0.18±0.12c
924
7.83±0.12a
6.92±0.93a
ND
ND
ND
ND
109
4.73±0.40a
4.19±0.38a
0.45±0.12b
ND
ND
ND
1.41±0.03b
2.21±0.29a
ND
ND
ND
ND
3-Thiophenecarboxylic
125
acid
7
Total carboxylic acid 3
Hexanoic acid, methyl
16
Benzoic acid, methyl ester
0
20
Octanoic acid, methyl
112
ester
3
Methyl nicotinate
113
ester
7
Nonanoic acid, methyl
122
ester
3
Cyclopentanecarboxyli
122
c acid, 2-pentyl ester
8
Butanoic acid, hexyl
136
ester
5
53
Hexanoic acid, hexyl
138
ester
3
55
Benzoic acid, 2-
143
52
methylbutyl ester
2
60
Dodecanoic acid,
152
methyl ester
2
61
Pentanoic acid, 2,2,4-
158
trimethyl-3-
2
0.73±0.06b
1.08±0.04a
0.34±0.02c
ND
ND
ND
ND
ND
1.14±0.09c
1.21±0.03bc
1.75±0.14a
1.33±0.12abc
1.59±0.09ab
0.77±0.03a
0.71±0.10a
0.65±0.23ab
0.34±0.04bc
0.32±0.03bc
0.19±0.01a
ND
0.12±0.01b
0.10±0.02bc
ND
ND
0.81±0.06a
0.49±0.05b
1.96±0.23a
0.84±0.07b
0.93±0.30a
b
9,12-Octadecadienoic
209
acid (Z,Z)-, methyl
1
73.89±17.38a
EP
2
1.58±0.11b
AC C
1.63±0.07b
16.08±5.25a
9.09±2.51a
1.27±0.92a
0.51±0.33d
MS
--
0.27±0.07c
MS
Green, waxy, soapy and fruity
0.07±0.01c
ND
RI,
Green, sweet, fruity, tropical
0.52±0.05b
0.69±0.11ab
0.18±0.11c
MS
--
ND
ND
ND
ND
RI,
Oily, wine, fruity, floral
0.57±0.06c
0.33±0.10c
0.26±0.06c
0.53±0.10bc
0.41±0.09b
0.18±0.07b
0.23±0.03b
0.05±0.00b
ND
ND
ND
ND
0.05±0.00b
ND
ND
6.60±0.27bc
3.67±0.26bc
4.21±0.60bc
2.75±1.54c
0.234±0.11
33.83±1.35
122.16±27.25
b
a
ND
ND
ND
10
2(3H)-Furanone, 5-
104
0.80±0.05
ND
ND
ND
ND
ND
ethyldihydro-
7
2-Decanone
119
2
Pyrazine, 2,5-dimethyl-
913
--
RI,
--
RI,
Odourless
MS
--
MS RI,
Sweet, creamy, lactonic, tobacco
MS 0.11±0.01cd
0.10±0.01d
0.15±0.01cd
0.94±0.17bc
1.46±0.07ab
2.25±0.73a
1
3
RI,
MS
ND
Total ketone
MS
MS
0.49±0.03a
dihydro-5-pentyl-
MS
MS
0.47±0.07a
135
Sweet, wintergreen, fruity
Sweet, fruity, waxy, tropical, wine
971
2(3H)-Furanone,
RI,
MS
4-Octanone
50
Warm, herbal, tobacco
ND
4
27
RI,
b
7
Total ester
1.27±0.20a
ND
methyl-, methyl ester
methyl ester, (E)-
0.73±0.15ab
3.61±0.33a
10.03±0.36
209
1.18±0.14ab
2.45±0.13b
192
9-Octadecenoic acid,
Green, fruity, waxy, citrus, fatty
MS
Pentadecanoic acid, 14-
ester 64
0.29±0.12ab
ND
isobutyl ester
63
Phenolic, cherry
MS
carboxyisopropyl,
62
RI,
SC
ester
RI,
MS
M AN U
116
33
Fruity, pineapple, ether
MS
TE D
Benzoic acid, ethyl
32
RI, MS
3 24
--
RI PT
21
MS
RI,
Orange, floral, fatty, peach
MS 2.98±0.12a
1.05±0.09b
0.89±0.08b
0.51±0.11b
0.61±0.11b
0.80±0.44b
4.36±0.21a
1.64±0.11c
1.04±0.08c
1.44±0.20c
2.07±0.04bc
3.05±1.17ab
ND
ND
ND
2.04±0.18b
4.95±0.10ab
6.57±3.43a
RI,
Sweet, coconut, coumarin, creamy
MS
RI,
Roasted, Nutty, peanut, musty
MS 14
Pyrazine, 2-ethyl-3,5-
107
ND
ND
ND
0.91±0.12b
28
6.48±0.07a
7.88±4.45a
RI,
Roasted, nutty
15
dimethyl-
3
Pyrazine, tetramethyl-
108
ACCEPTED MANUSCRIPT
MS
ND
ND
ND
0.11±0.00b
0.70±0.18ab
1.05±0.59a
ND
ND
ND
3.06±0.29bB
12.13±0.28ab
15.50±8.46a
RI,
Roasted
MS
0 Total pyrazine
A
7
1,2-Benzenediamine, 4-
100
methyl-
2
Total
ND
ND
ND
1.28±0.08b
5.83±0.15a
7.45±3.72a
ND
ND
ND
1.28±0.08b
5.83±0.15a
7.45±3.72a
11.07±0.73
12.34±1.40a
13.95±1.62
12.49±1.86a
9.84±1.53a
2.61±1.76b
47.26±4.42b
59.53±4.42b
44.25±27.43
MS
--
a
Total
a
81.29±3.25
161.72±26.42
52.71±4.60
b
a
b
b
RI PT
phenylenediamine Total unknown ****
* RI = retention index
572
** ID = Identification by comparison of mass spectra (MS) with in-house NIST library 11.0, and
573
MS and retention index (RI) of NIST Webbook (NIST, 2017).
574
*** = Aroma descriptions were obtained from Acree and Arn (2017) and TGSC (2017)
575
**** = Sum of all the unidentifiable compounds (peak No. 8, 22, 26, 34, 35, 38-45, 48, 49, 51, 56,
576
57).
577
ND = Not detected or value below the detection threshold.
578
-- = Not available.
579
a-f
580
A-C
581
and values with different superscripts in the same row are significantly different (P ≤ 0.5)
TE D
M AN U
SC
571
= Values with different superscripts in the same row are significantly different (P ≤ 0.05).
AC C
EP
= Additional one-way ANOVA test was performed to remove the large error grain infusion data,
29
ACCEPTED MANUSCRIPT
Highlight: Sorghum grain was soaked, steamed, and roasted to make grain tea.
•
Processing techniques affected total phenolic content but not antioxidant activity.
•
A total of 63 volatile compounds were detected in sorghum grain tea.
•
Main volatile compounds were alcohols, alkanes, aldehydes, and esters.
•
More volatile compounds were extracted in sorghum powder than whole grain infusion.
AC C
EP
TE D
M AN U
SC
RI PT
•