Hydrolysis before Stir-Frying Increases the Isothiocyanate Content of

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Hydrolysis before stir-frying increases the isothiocyanate content of broccoli Yuanfeng Wu, Yuke Shen, Xuping Wu, Ye Zhu, Jothame Mupunga, Wenna Bao, Jun Huang, Jianwei Mao, Shiwang Liu, and Yuru You J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05913 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Journal of Agricultural and Food Chemistry

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Hydrolysis before Stir-frying Increases the Isothiocyanate Content of Broccoli

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Yuanfeng Wu*,†,§, Yuke Shen†,#, Xuping Wu†,#, Ye Zhu†,#, Jothame Mupunga†,#,

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Wenna Bao†,#, Jun Huang†,#, Jianwei Mao†,#, Shiwang Liu†,#, and Yuru You†,#

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† School of Biological and Chemical Engineering, Zhejiang University of Science

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and Technology, Hangzhou, 310023, Zhejiang, China

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§ Guangdong Province Key Laboratory for Green Processing of Natural Products

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and Product Safety, Guangzhou, 510640, Guangdong, China

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# Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm

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Produces, Hangzhou, 310023, Zhejiang, China

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*Corresponding author, Tel: +86-571-85070396, Fax: +86-571-85070378

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Email: [email protected];

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E-mail for all co-authors:

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Wu Xuping: [email protected];

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Zhu Ye: [email protected];

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Shen Yuke: [email protected];

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Jothame Mupunga: [email protected];

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Bao Wenna: [email protected];

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Huang Jun: [email protected];

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Mao Jianwei: [email protected];

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Liu Shiwang: [email protected];

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You Yuru: [email protected]

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ACS Paragon Plus Environment


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Abstract: Broccoli is found to be a good source of glucosinolates, which can be

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hydrolyzed by endogenous myrosinase to get chemopreventive isothiocyanates (ITCs),

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among them sulforaphane (SF) is the most concerned agent. Studies have shown that

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cooking greatly affected the levels of SF and total ITCs in broccoli. However, little is

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documented about the stability of these compounds during cooking. In this study, we

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proved that the half-lives of SF and total ITCs during stir frying were 7.7 and 5.9 min,

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respectively, while the myrosinase activity decreased by 80% after 3 min of stir-frying,

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SF and total ITCs were more stable than myrosinase. Thus, the contents of SF and

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total ITCs decreased during stir-frying largely because myrosinase was destroyed.

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Subsequently, it was confirmed that compared to direct stir-fry, hydrolysis of

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glucosinolates in broccoli for 90 min and followed by stir-frying increased the SF and

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total ITCs concentration by 2.8 and 2.6 times, respectively. This method provides high

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quantities of beneficial ITCs even after cooking.

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Keywords: Broccoli, stir-frying, sulforaphane, isothiocyanate, myrosinase

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Introduction

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Broccoli has been widely approved for its beneficial effects on human health because

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it contains high concentrations of vitamins, minerals, polyphenols and in particular a

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group of phytochemicals named glucosinolates1. Epidemiological studies have shown

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that broccoli has potential beneficial effects, including the prevention or reversal of

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cancer2, hepatic steatosis3, and cardiovascular disease4. The health effects of broccoli

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are related to its high content of glucosinolates5. When fresh broccoli florets or

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broccoli sprouts are crushed, chopped or chewed, glucosinolates are hydrolyzed to

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equimolar amounts of glucose and unstable thiono compounds. The latter are

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spontaneously converted into bioactive isothiocyanates (ITCs) by the action of an

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endogenous enzyme called myrosinase, or non-bioactive nitriles by the action of

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epithiospecifier protein (ESP)5,6. The most abundant glucosinolate found in broccoli is

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glucoraphanin, followed by glucobrassicin, which are hydrolyzed to sulforaphane (SF)

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and indole-3-carbinol by the action of myrosinase7.

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Before consumption, broccoli is commonly cooked. Cooking methods such as

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steaming and microwaving are common in Western society, whereas stir-frying is the

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most popular method in China8. SF has many bioactivities, and methods to retain or

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improve its content in cooked broccoli are vital. Some studies have shown that

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cooking methods greatly alter the content of total glucosinolates and their

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hydrolysates, as well as the myrosinase activity in brassica vegetables9. For example,

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steaming broccoli for 2 min has a significant inhibitory effect on SF production10. A

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study by Rungapamestry et al.11 showed that hydrolysis of glucoraphanin to SF and



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absorption of SF in humans after consumption of lightly-cooked broccoli

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(microwaved for 2 min) were about three times those after consumption of

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fully-cooked broccoli (microwaved for 5.5 min), indicating that a longer cooking time

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inhibits SF production. Jones et al.9 reported that SF production decreased from 51.9

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to less than 1.1 mg/kg dry weight (DW) after microwaving or boiling for 2-5 min, and

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steaming for 2 min resulted in SF yield loss of about 50%. Therefore, the authors

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suggested that for optimum SF intake, broccoli should be eaten raw or lightly steamed.

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Vermeulen et al.12 found that, compared with microwaved broccoli, raw broccoli

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consumption led to much higher SF levels in urine and blood. SF was not detectable

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in broccoli florets after boiling for 15 min or steaming for 23 min, whereas

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pressure/microwave cooking for 2 min only caused 17% loss of SF13. Generally,

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compared with raw broccoli hydrolyzed by endogenous myrosinase during digestion,

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intake of cooked broccoli usually provides approximately one tenth of the amount of

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SF14-16. These results show that only raw or lightly processed broccoli provides high

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levels of beneficial SF.

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The stability of SF under different conditions and in different systems has been

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reported in a number of studies17,18. As an inherently reactive small molecule, SF is

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reported to be thermally sensitive and readily degradable17,18. Surprisingly, few

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methods have reported the SF concentrations in stir-fried broccoli, and to the best of

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our knowledge, no report has focused on SF stability in the stir-frying process. In a

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previous study, we showed that the half-life (t1/2) of SF at 90°C and pH 6.0 was 1.5

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h19. Considering that the stir-frying time is usually 4 min, SF and total ITCs may not


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be as labile as we thought. Nevertheless, myrosinase is very labile, which in broccoli

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tissue and juice were stable up to 45°C and 40°C, respectively20,21. Consequently, we

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expect that since myrosinase is much more labile than ITCs, myrosinase will be

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destroyed during cooking, and formation of SF and total ITCs will greatly decrease.

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Thus, allowing for hydrolysis before stir-frying should increase the SF and total ITCs

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contents in broccoli.

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In this study, we evaluated the stabilities of SF, total ITCs, and myrosinase during

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stir-frying process. A method for modifying the broccoli stir-frying process to increase

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SF and total ITCs formation is proposed.

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Materials and Methods

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Materials

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Broccoli seeds, namely LS-1, were preserved at Zhejiang Provincial Key Lab for

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Chem & Bio Processing Technology of Farm Products. Broccoli and carrots were

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purchased from a local market. One single cultivar of broccoli purchased at one time

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was used to study myrosinase activity, and another single cultivar of broccoli

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purchased at another time was used to study ITCs contents before and after stir-frying.

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Sulforaphane (98%) and sinigrin hydrate (99%) were purchased from Sigma

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Chemical Co. (St. Louis, MO, USA), 1,2-Benzenedithiol was purchased from TCI Co.

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(Tokyo, Japan), Glucose GOD/PAP Kit was purchased from Nanjing Jiancheng

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Biotech Inc. (Jiangsu, China), and Protein Quantitation Kit was purchased from

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Sangon Biotech (Shanghai, China). Methanol (Merck, USA) was HPLC grade, and all

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other chemicals used were of analytical grade and purchased either from Aladdin



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(Shanghai, China) or Sangon Biotech (Shanghai, China).

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Total ITCs extraction methods

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Total ITCs was extracted by the method previously reported19. Briefly, 50 g broccoli

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seed meal was ground using a Chinese herbal medicine grinder for 15 s. 400 ml of

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hexane was added into the seed meal and stirred for 3 h. The seed meal was then

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transferred and dried in a fume hood overnight to obtain de-fatted seed meal. In the

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hydrolysis and extraction step, 200 ml ethyl acetate and 100 ml potassium phosphate

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buffer (0.05 M, pH 5.8) were added simultaneously into the seed meal and was

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agitated for 4 h, then 20g sodium chloride and 10 g anhydrous sodium sulfate were

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added to the solution and agitated. The ethyl acetate layer was transferred and the

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residual seed meal was extracted twice by equal volume of ethyl acetate, the resulting

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extracts were combined and dried in a rotary evaporator (Yarong Instrument,

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Shanghai, China). The residue was diluted with water and stored in a refrigerator at

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-20°C.

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Stability of SF and total ITCs during stir-frying

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100g of carrots were cut into 0.3 cm dice, and 10 ml broccoli extracts with SF

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concentration of 4.17 mg/ml was added and mixed. An electromagnetic oven with

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output power of 1200 W was preheated for 1 min, then carrots and broccoli extracts

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mixture were stir-fried in a wok, at 0, 1, 2, 3, 4, 6, 8 and 10 min, 5 g sample was taken

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and was rapidly cooled by placing them in an ice water bath. Each sample was

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extracted by 15 ml ethyl acetate for three times, and the resulting ethyl acetate layers

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were combined and evaporated at 30 °C. The residue was dissolved by methanol and


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constant-volumed to 10 ml. The SF and total ITCs concentrations of each sample

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were measured by HPLC, as described below. Three independent replicates were

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carried out for each experiment.

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Determination of myrosinase activity during stir-frying

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Myrosinase activity was measured by the method of Guo et al.22 with some

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modifications. Briefly, an electromagnetic oven with the output power of 1200 W was

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preheated for 1 min, then a total of 100g broccoli were stir-fried in a wok, as

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described above. At 0, 1, 2, 3, 4, 6 and 8 min, 5 g sample was taken and was cooled

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down by placing them into an ice water bath. Each sample was divided into two equal

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parts, one part was heated at 80 °C overnight to obtain dry weight. Another part was

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homogenized with 15 ml 0.1 M phosphate buffer (pH 6.5) in an ice bath for 3 min.

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The homogenate was centrifuged at 10000 g and 4°C for 15 min. 500 µl supernatant

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was mixed with 500 µl distilled water and boiled for 5 min, then the amount of

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glucose in the mixture (A1) was quantified using glucose GOD/PAP kit (Nanjing

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Jiancheng Biotech Inc., Jiangsu, China). Another 500 µl supernatant was mixed with

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500 µl 0.25 mM sinigrin solution, and incubated at 37 °C for 15 min. The reaction

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was stopped by boiling for 5 min. The amount of glucose in the mixture (A2) was

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analyzed using the glucose GOD/PAP kit. The glucose formed in the enzymatic

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hydrolysis was calculated by subtracting A1 from A2. One myrosinase unit was

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expressed as 1 nmol glucose formed per min. The specific activity was defined as

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units per gram of broccoli (dry weight). Three independent replicates were carried out

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for each experiment.



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The Hydrolysis rate of myrosinase

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2.5g broccoli were homogenized with 15 ml 0.1 M phosphate buffer (pH 6.5) in an ice

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bath for 3 min. The homogenate was centrifuged at 10000 g and 4°C for 15 min to get

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the supernatant. 0.25 ml of 2.5 mM sinigrin were mixed with 0.25 ml supernatant,

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well capped and incubated at 37 °C. At 0, 10, 15, 30, 45, 60, 90, 120, 150 and 210

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min intervals, one tube was randomly taken from the water bath, and boiled for 5 min

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to quench the reaction. The amount of glucose was measured using the glucose

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GOD/PAP kit. Three independent replicates were carried out for each experiment.

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The effect of hydrolysis on ITCs content after stir-frying

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Fresh broccoli florets were cut into small pieces (size approximately 0.2 × 0.2 × 0.2

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cm) and were divided into three groups, namely hydrolyzed and stir-fried (HS) group,

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directly stir-fried (DS) group and raw broccoli (RB) group. For the HS group, 100 g

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of chopped fresh broccoli were mixed with 3 ml water and incubated in a 37°C water

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bath for 90 min. An electromagnetic oven with the output power of 1200 W was

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preheated for 1 min, and the broccoli was stir-fried in a wok for 4 min. After which

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the broccoli meal was mixed with 200 ml ethyl acetate and 100 ml 0.05 M potassium

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phosphate buffer (pH 7.0), agitated for 30 min, 25 g NaCl were then added and the

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mixture was shaken and filtered by filter paper. The ethyl acetate layer was transferred,

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the water layer was extracted by 200 ml ethyl acetate for another 2 times. The ethyl

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acetate layers were combined and evaporated. The residue was dissolved using 25 ml

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methanol. For the DS group, the electromagnetic oven with output power of 1200 W

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was preheated for 1 min, 100 g of chopped broccoli florets were stir-fried for 4 min,


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then the broccoli florets were rapidly cooled down by placing them in an ice water

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bath. The florets were mixed with 200 ml ethyl acetate and 100 ml potassium

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phosphate buffer, and the proceeding steps were as same as those done on the HS

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group. For the RB group, 100 g of chopped fresh broccoli was mixed with 200 ml

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ethyl acetate and 100 ml 0.05 M potassium phosphate buffer (pH 7.0), and the

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proceeding extraction steps were also as the same as those done on the HS group.

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Three independent replicates were carried out for each condition, Total ITCs

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concentration in the methanol solution was measured by HPLC, individual ITCs,

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nitriles and degradation products were determined by GC-MS, as described below.

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Total ITCs Quantification

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Total ITCs quantification was performed using the cyclocondensation method23.

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Briefly, ITCs sample was mixed with 10 mM of 1,2-benzenedithiol and 25 mM of

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potassium phosphate buffer. After incubated for 2 h at 65°C, the mixture was cooled

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down to room temperature and then centrifuged at 16000 g for 10 min. The

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supernatant was collected and analyzed using a Waters e2695 HPLC System (Milford,

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MA, USA) equipped with a 4.6×250 mm i.d., 5 µm WondaCract ODS-2 column

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(Shimadzu, Japan). The mobile phase consisted of 80% methanol and 20% water at a

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flow rate of 1.0 ml/min, and 10 µl sample was injected onto the column. The

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cyclocondensation product, 1,3-benzodithiole-2-thione, was detected by absorption at

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wavelength of 365 nm using a Waters 2489 detector (Milford, MA, USA). Standard

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curve was prepared using varying amounts of pure SF to estimate the ITCs

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concentration in each sample.



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SF quantification

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SF was quantified using a Waters e2695 HPLC system (Milford, MA, USA) by the

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method previously reported19. 10 microliters of sample were injected onto a 4.6×250

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mm i.d., 5 µm WondaCract ODS-2 column (Shimadzu, Japan). The mobile phase

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consisted of methanol and water, the flow rate was 1 ml/min, and the column oven

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temperature was 25 °C. Gradient conditions: Methanol was increased from 20% in

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water to 60% over 10 min period, then increased to 100% in 2 min and maintained for

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another 2 min. SF was detected by absorbance at 241 nm using a Waters 2489

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detector (Milford, MA, USA).

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GC-MS analysis

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Glucosinolate hydrolysis products were determined by a 7890A GC System (Agilent

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Technologies Inc, USA) by the method previously reported24. Mass spectra were

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obtained by an 5975C inert XL/CI MSD with Triple-Axis Detector enabling recording

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of ions from m/z 35 to m/z 500. The capillary column was an HP-5MS UI capillaries

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(0.25 μm film thickness, 30 m × 0.25 mm i.d.). The injection volume was 1.0 µL.

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The injector and detector temperature were set at 270 and 280°C, respectively. The

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column oven temperature was began at 50°C for 2 min, increased at 10 °C/min to

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190 °C, then increased at 20 °C/min to 300 °C, and was maintained for 5 min. The

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carrier gas was ultra-high purity helium at flow rate of 1 ml/min, and the split ratio

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was 10:1. Cyclohexanone was used as the internal standard.

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Statistical Analysis

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All experiments were done in triplicates. Treatment effects were determined by


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analysis of variance (one-way ANOVA) using SPSS 22.0 software (IBM Corporation,

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NY, USA). Differences were considered significant at p values0.9, showing that the

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degradation of SF and total ITCs during stir-frying followed first order reaction

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kinetics. The t1/2 of SF and total ITCs were calculated by linear regression of the

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observed data in Figure 2, and were 7.7 min (SF) and 5.9 min (total ITCs). In our

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previous study, the t1/2 of SF in a pH 6.0 solution at 90°C was 1.5 h19, much longer

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than that observed in this study. This is reasonable because SF was not in solution in

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the present study and the temperature was higher than 90°C. Considering that the


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stir-frying time is always much shorter than 10 min, our results indicate that SF and

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total ITCs are somewhat stable during stir-frying.

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Myrosinase stability and activity during stir-frying

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The stability of myrosinase during stir-frying was studied. Fresh broccoli was

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chopped, stir-fried for 8 min, and the myrosinase activity at 0, 1, 2, 4, 6 and 8 min

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were measured (Figure 3). Based on sinigrin hydrolysis measured with a glucose

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assay kit, the myrosinase activity of raw broccoli was 128 U/g DW broccoli. Guo et

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al.22 reported a myrosinase activity of 75.2 U/mg protein. Okunade et al.29 reported

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that the myrosinase activity of brown mustard was the highest (2.04 un/mg) whilst

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that of yellow mustard was the lowest (0.48un/mg). The variations in myrosinase

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activity within and between Brassica species have been attributed to genetic and/or

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environmental factors (agronomic and climatic conditions)30 as well as different

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enzyme activity definitions.

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Myrosinase activity decreased by 80% after stir-frying for 3 min (Figure 3). This

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result is not unexpected because myrosinase has been reported to be easily inactivated

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at normal cooking temperatures, regardless of the cooking method used31. Myrosinase

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in Brassicaceae lost approximately 90% of its activity after heating at 60°C for only 3

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min32. Microwave cooking of red cabbage for 4.8 min at 900 W caused almost

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complete loss of myrosinase activity33. Gliszczyńska-Świgło et al.34 reported that the

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black mustard enzyme activity in cabbage was lost after 2 min of microwave cooking

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or 7 min of steaming. Compared to other myrosinase sources, broccoli myrosinase has

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a low thermal stability35, and heating broccoli florets to 70°C or above for 5 min



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inactivates the myrosinase36. Generally, myrosinase is temperature sensitive and

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cooking methods such as steaming or microwaving are known to decrease or

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completely loss of the hydrolytic activity.

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Furthermore, the enzyme-hydrolysis rate of glucosinolates was studied. The result

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showed that the hydrolysis rate was very slow. In the first 60 min, the glucose content

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increased very slowly. After 90 min, the glucose content increased to a certain extent

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(0.34 µM) and then level off with further increases (Figure 4). This result was in line

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with earlier studies. Tian et al.37 studied the effect of the hydrolysis time on SF

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production from broccoli sprouts, and found that the best hydrolysis time for SF

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production was 1.5 h. Vastenhout et al.38 evaluated the kinetics of glucosinolate

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hydrolysis by Sinapis alba myrosinase, found that production of ITCs increased

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gradually in 80 min. Similar results were reported by Klingaman et al.39 using another

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glucosinolate. The results for these reports were different, because the hydrolysis rate

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is dependent on the structure of the ITC25. However, from these results, we could infer

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that the hydrolysis rate is very slow.

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In summary, the enzymatic activity study showed that myrosinase was labile, and that

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the cooking procedure inactivated myrosinase. The hydrolysis rate is very slow,

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therefore during cutting or mincing of broccoli, only a small quantity of the

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glucosinolates are hydrolyzed to ITCs.

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The effect of hydrolysis on the ITCs content after stir-frying

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Based on the above results, we hypothesized that hydrolysis of glucosinolates in

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broccoli by endogenous myrosinase for a certain length of time (e.g., 90 min) before


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stir-frying, the nutritional value would then increase because ITCs are more stable

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than myrosinase. To verify this, the total and individual ITCs contents in the

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hydrolyzed and stir-fried (HS) group, directly stir-fried (DS) group and raw broccoli

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(RB) group were analyzed. The total ITCs concentration in broccoli was quantified by

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the cyclocondensation method, which is both accurate and rapid23, and individual

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ITCs and nitriles were determined by GC-MS. The cutting style greatly affected the

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ITCs content in broccoli, and the total ITCs concentration increased by 133% in

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florets processed into chops compared to other cutting styles40. Thus, we processed

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broccoli florets into chops, and waited for 90 min before stir-frying to allow for

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production of as much ITCs as possible. During stir-frying, broccoli is usually cooked

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for about 4 min, therefore stir-frying of 4 min was used in this study. The results

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verified our hypothesis, the ITCs formation in the RB, HS and DS groups were

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37.92±13.35, 30.90±10.88 and 11.89±2.52 mg/(100g DW broccoli), respectively

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(Figure 5). The ITCs production in RB and HS groups were higher than DS group, as

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compared to DS group, the ITCs content in HS group increased by 2.6 folds, while

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there was no significant difference between RB and HS groups.

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Volatile compounds from the methylene chloride extracts of the RB, HS and DS

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groups, such as ITCs, nitriles and their degradation products, were identified and

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quantified by GC-MS (Table 1). SF was the most abundant ITCs, followed by

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1-butene 4-isothiocyanate and erucin, only trace amounts of PEITC were detected in

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the RB group. After stir-frying, there were no significant differences of SF and erucin

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contents between RB and HS groups, while in the DS group, SF content decreased



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significantly, and only trace amounts of erucin were found in the DS group.

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Some degradation products of SF were also found. Among them, dimethyl sulfone,

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S-methyl methylthiosulfinate, S-methyl methylthiosulfonate have been reported as

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thermal degradation products of SF, and the mechanisms for the formation of these

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compounds from SF have been proposed by Jin et al18. Concentration of these

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degradation products was higher in the RB group but much lower in the HS and DS

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groups. We supposed that these compounds are volatile and volatilized in the

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stir-frying procedure.

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Sulforaphane nitrile and 3-methyl-2-butenenitrile, produced in the action of

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epithiospecifier proteins (ESP), were also found. ESP is the cofactor of myrosinase,

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which can direct the hydrolysis reaction towards nitriles in the presence of Fe2+ ions36.

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Unlike ITCs, nitriles are much lower in the DS and HS groups. For example,

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3-methyl-2-butenenitrile was not present in the DS and HS groups, but in the RB

337

group, its concentration was 5.43±1.59 mg/100 g DW broccoli. There were no

338

significant differences between the concentrations of sulforaphane nitrile in the HS

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and DS groups, but in the RB group, the concentration was higher. Nitriles are much

340

stable than ITCs, which can present even during heat treatments at temperature higher

341

than 100°C and hardly degrade any further26. Therefore, their losses during stir-frying

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predominantly occur because of their volatility.

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A significant increase (p

Hydrolysis before Stir-Frying Increases the Isothiocyanate Content of

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