Keywords

1 Introduction

Broccoli (Brassica oleracea L. var. italica) is an economically important vegetable crop in many countries and has been highly valued by consumers due to its flavor as well as its nutritional components like minerals, vitamins, polyphenols, and other beneficial phytochemicals, particularly potent anticarcinogenic glucosinolates (GSs) [1–4]. Epidemiological studies have suggested that the consumption of broccoli could lower the risk for the development of certain forms of cancers, which have been attributed to glucosinolates and their degradation products [5, 6].

Glucosinolates , a group of sulfur- and nitrogen-containing secondary metabolites , mainly belong to the Brassicaceae family. The general structure of glucosinolate consists of a β-d-thioglucose group, a sulfonated oxime group, and a side chain derived from amino acids [7]. More than 200 glucosinolates have been identified so far [8] and can be divided into three classes on the basis of their derived amino acid precursors: aliphatic glucosinolates, indole glucosinolates, and aromatic glucosinolates. Usually, glucosinolates with different structures can be degraded into various biologically active breakdown products in broken tissues and living cells of plant, as well as gastrointestinal tract of mammalian [9–14], which contribute to the flavor, anticarcinogenic activity, and resistance of broccoli [15]. As maintaining and enhancing good health through dietary habits have been proposed to deal with increased lifestyle diseases in current society, optimal glucosinolate profile and content in broccoli are expected. The purpose of this chapter is to review the research findings related to glucosinolate biosynthetic pathway and variation among different broccoli genotypes, as well as the regulation of glucosinolate accumulation along the whole food chain for better retention of glucosinolates in broccoli.

2 Biosynthetic Pathway of Glucosinolates in Broccoli and Brassica Plants

So far, the biosynthetic pathway of glucosinolates has been successfully elucidated in Arabidopsis and summarized in several review papers [7, 16]. Generally, aliphatic glucosinolate biosynthesis consists of three separate steps: (i) chain elongation of precursor amino acid, (ii) development of the core glucosinolate structure, and (iii) secondary modifications of the amino acid side chains [7, 16, 17]. There are five reactions required for precursor amino acid elongation: an initial and final transamination, acetyl-CoA condensation, isomerization, and oxidative decarboxylation [18, 19]. In this process, branched-chain amino acid aminotransferase (BCAT), bile acid transporter 5 (BAT5), methylthioalkylmalate synthase (MAM), isopropylmalate isomerase (IPMI), and isopropylmalate dehydrogenase (IPMDH) are involved [20–26]. The core glucosinolate structure formation begins with oxidation of precursor amino acids by cytochrome P450 monooxygenases (cytochrome P450s) of the CYP79 family and then CYP83 family, followed by further metabolic process catalyzed by C-S lyase, glucosyltransferase, and sulfotransferase [27–35]. The secondary modifications are attributed to several loci, including GS-OX which catalyzes methylthioalkyl to methylsulfinylalkyl glucosinolates, GS-ALK/AOP2 which controls the conversion to alkenyl glucosinolates, GS-OHP/AOP3 which is responsible for the production of 3-hydroxypropyl glucosinolates, and GS-OH which participates in the production of 2-hydroxy-3-butenyl glucosinolate in aliphatic glucosinolate biosynthesis [36–38], as well as cytochrome P450 CYP81Fs and indole glucosinolate methyltransferase (IGMT) in indole glucosinolate biosynthesis [39–42]. Furthermore, several regulators of glucosinolate biosynthesis have also been identified, such as nuclear-localized calmodulin-binding protein IQD1, DOF transcription factor AtDof1.1, sulfur limitation1 (SLIM1), TU8, and six R2R3-MYB transcription factors as well as three basic helix-loop-helix (bHLH) transcription factors [43–54].

As the genome sequence of Brassica crops including B. oleracea has been reported [55], genes related to glucosinolate biosynthesis in Brassica crops were identified gradually with the assistance of bioinformatics. The aliphatic glucosinolate biosynthesis is described in Fig. 1 based on previous studies and genome sequence. BoGSL-ELONG, BoGSL-ELONGL, BoGSL-PRO, and BoGSL-PROL, which control chain elongation of precursor amino acid in B. oleracea, have been cloned or inferred [56–59]. Li and Quiros [56] have conducted the genetic analysis, expression, and molecular characterization of BoGS-ELONG, which was responsible for the synthesis of four-carbon glucosinolates in B. oleracea. However, BoGS-ELONGL, which corresponds to MAM-L in Arabidopsis, was probably nonfunctional, at least in broccoli variety ‘Early Big’ [58]. In addition, genetic analysis suggested that BoGS-PRO was associated with the biosynthesis of three-carbon side-chain glucosinolates [57], while the function of BoGS-PROL has not been assessed yet [59]. BoGS-ALK was also cloned and demonstrated to be responsible for alkenylation in side-chain modification in glucosinolate pathway [57]. Furthermore, BoCYP79F1, BoCS-lyase, BoS-GT (S-glucosyltransferase), BoGS-OH, and the candidate gene for BoGS-OXID have also been inferred [60, 61].

Fig. 1
figure 1

Aliphatic glucosinolate biosynthesis in Brassica oleracea

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Although Brassica crops share the similar pathway of glucosinolate biosynthesis with Arabidopsis, the genes involved are different in sequence identities with their counterparts in Arabidopsis as well as with other Brassica crops. For instance, MAM family contains high copies in B. rapa and B. oleracea, however, with different expression patterns, resulting in the fact that the major aliphatic glucosinolates are 3C and 4C glucosinolates in B. oleracea while 4C and 5C glucosinolates in B. rapa [55].

3 Genetic Variation and Function of Glucosinolate Profiles in Broccoli

3.1 Genetic Variation of Glucosinolate Composition and Content

According to previous reports, there are up to 17 different kinds of glucosinolates identified in broccoli (Table 1) with a genetic variation in their composition and content [62–65]. Nevertheless, glucoraphanin is the predominant methionine-derived aliphatic glucosinolate in most varieties. In an early report, Carlson et al. [62] determined the variation of nine glucosinolate profiles in six commercial cultivars and found that glucoraphanin contents were from 29.2 to 88.3 μmol 100 g−1 fresh weight (FW). In 1999, Kushad et al. [64] identified 14 glucosinolates in 50 broccoli accessions, and glucoraphanin levels were in a scope from 0.8 to 21.7 μmol g−1 dry weight (DW). Similarly, Farnham et al. [66] demonstrated that the amount of glucoraphanin varied from 0.04 to 2.94 μmol g−1 FW in 71 pure lines and 5 hybrid checks. The results from Chinese broccoli germplasms indicated that the glucoraphanin concentrations ranged from 0.06 to 24.17 μmol g−1 DW in 143 pure lines and from 1.57 to 5.95 μmol g−1 DW in 5 commercial varieties [65], while progoitrin levels in 5 commercial cultivars differed from 1.77 to 6.07 μmol g−1 with a mean of 3.20 μmol g−1 DW. However, reports about high levels of progoitrin or epiprogoitrin were few in primary broccoli cultivars from America [62, 64, 67], England [68], Germany [69], Portugal [70], and Spain [71]. Likewise, relatively higher concentration and variation in indole glucosinolate were reported by most surveys [65, 68–71] except for Kushad et al. [64]. Generally, the content of aromatic glucosinolates, mainly gluconasturtiin, is much lower than aliphatic or indole group. Moreover, some specific lines with altered glucosinolate profiles were observed in broccoli germplasms [65]. Former studies have demonstrated that the accumulation of aliphatic glucosinolates in broccoli head is mainly regulated by genetic factors, while the content of indole glucosinolates is greatly affected by environment and environment × genotype interaction [63, 85, 86]. Recently, the identification of two glucosinolate transporters, GTR1 and GTR2, which are response for the transportation of glucosinolates in Arabidopsis, provided us a useful means to control the allocation of glucosinolates in different tissues [87].

Table 1 Trivial and chemical name of glucosinolates identified in broccoli
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The breakdown pathway as well as products of glucosinolates is a leading area in glucosinolate research in recent years. The classical breakdown pathway in plant depends on classic myrosinase (β-thioglucoside glucohydrolase, TGG) [17], which is localized in separate plant cells or in separate intracellular compartments with glucosinolates in intact plant tissue while tissue damage brings them together and initiates TGG-catalyzed glucosinolate hydrolysis [88, 89]. In addition, an atypical myrosinase, PENETRATION2 (PEN2), is thought to be involved in a new indole glucosinolate catabolic pathway in living plant cell [39, 40, 90]. Furthermore, intact glucosinolates could also been degraded by the resident microflora of the gastrointestinal tract to form ITCs, amines, or nitriles depending on the type of bacterial myrosinase-like activity involved [10–14].

In Arabidopsis, there are six myrosinase genes with different gene sequences and expression patterns [9]. Besides, some specific proteins , such as epithiospecifier protein (ESP) and nitrile-specifier protein (NSP), are also involved in the hydrolysis of glucosinolates, affecting the final breakdown products. Mithen et al. [91] compared the ability to induce phase II detoxification enzymes in two broccoli lines at similar glucosinolate level with ‘Marathon’, a standard commercial broccoli cultivar. They found one line showed 80 times the ability to induce quinone reductase compared to ‘Marathon’, while the other showed little induction. The detection of breakdown products showed that plenty of ITCs (95%) were degraded from glucoiberin and glucoraphanin, the predominant glucosinolates of broccoli, in the former line, while high levels of nitriles, and only 1% ITCs, were formed from glucoiberin and glucoraphanin in the later line. The results of sequences for B. oleracea sp. capitata indicated that the B. oleracea species also had a complex glucosinolate-myrosinase system [55]. In vitro, a recombinant broccoli ESP from cv. Packman directed myrosinase-dependent degradation of epiprogoitrin toward the formation of epithionitrile and glucoraphanin to sulforaphane nitrile instead of sulforaphane [92]. In addition, the variety of breakdown products from aliphatic glucosinolates, mainly glucoraphanin, in floret and sprout among different broccoli varieties was observed in some studies [91–93]. These researches indicated that the typical glucosinolate-myrosinase was much complex upon tissue damage, and genetic effect played an important role in hydrolysis of glucosinolates, especially aliphatic glucosinolates.

3.2 Biological Functions of Individual Glucosinolate Profiles

Glucosinolates and their hydrolysis products in broccoli have been considered to be bioactive, while some of them may have adverse effects for human and animal. There are in vitro and in vivo evidences that ITCs play multiple roles in cancer prevention. For example, some ITCs from methionine- and aromatic-derived glucosinolates such as sulforaphane, allyl isothiocyanate, and phenethyl isothiocyanate can strongly inhibit phase I enzymes and induce phase II enzymes, as well as promote cell cycle arrest and apoptosis in various cancer cell lines [94–100]. Indole-3-carbinol (I3C), a breakdown product from indole glucosinolate, has been found to exert chemoprotective activity through altering estrogen metabolism [101, 102]. Moreover, ITCs, notably sulforaphane, make contributions to cancer protection via inhibition of tumor invasion and angiogenesis [103], anti-inflammatory [104], as well as immunomodulatory activities [98]. In addition to these cancer chemopreventive properties, ITCs also play important roles in protection of the central nervous system [105], preventing against cardiovascular disease [106, 107] and bacterial infection [76].

As we have mentioned above, glucosinolates could be degraded to not only ITCs but also nitriles due to different hydrolytic environments. Previous research verified that quinone reductase and glutathione S-transferase activities in hepatic, colonic mucosal, and pancreatic were induced by high doses of sulforaphane but not by sulforaphane nitrile. Quinone reductase activity of sulforaphane and sulforaphane nitrile treated Hepa 1c1c7 cells also suggested that production of sulforaphane rather than sulforaphane nitrile could increase the potential chemoprotective effects of broccoli [108].

Glucosinolates and their breakdown products also show some adverse effects besides their healthy benefit functions. Indole derivatives (e.g., I3C) have been classified as bifunctional inducers [109, 110]. It has been reported that I3C inhibited the development of cancer in animals when given before or with a carcinogen, whereas enhanced development of cancer was observed when administered after a carcinogen in some cases [5]. Certain glucosinolates, such as sinigrin, progoitrin, and gluconapoleiferin, are responsible for bitter taste [111], which may decrease consumer acceptance. Furthermore, progoitrin has been considered as natural toxicants for its derivative has goitrogenic effects on mammals [112]. However, the amount of progoitrin in broccoli is quite low, and the occurrence of these substances is dependent on many cofactors. In addition, this malnutritional effect can also be avoided by normal iodine intake [113].

Glucosinolates and their hydrolytic products have been demonstrated to be crucial in plant defense response . They can stimulate feeding or oviposition by crucifer-specialist herbivores [16, 114–116]. Aliphatic glucosinolates were reported to be important for resistance of plants to pests [117], and sulforaphane functioned in nonhost resistance in Arabidopsis-Pseudomonas pathosystem [118]. Moreover, PEN2-dependent potential hydrolysis products of 4-methoxy glucobrassicin were found to activate innate immunity, leading to protection against fungal penetration [39, 40], while TGG-catalyzed degradation of indole glucosinolates attenuated mycotoxin fumonisin B1-induced programmed cell death in Arabidopsis [119].

4 Breeding of Broccoli Varieties with Optimal Glucosinolate Composition and Content

A high-glucoraphanin commercial hybrid, Beneforté®, was developed by Mithen’s group through genome introgression from the wild species Brassica villosa [120]. In former surveys of this group, a major quantitative trait locus (QTL) on linkage group 2 that determined the concentrations of methionine-derived glucosinolates in the high-glucoraphanin hybrids was found, and a microsatellite marker OI12-F02 originated from a wild species Brassica villosa was developed [68, 91]. Recently, researchers of this group cloned the major dominant QTL and identified an MYB28 allele resulted in higher expression of MYB28 in the leaves of high-glucoraphanin hybrids compared to the standard broccoli cultivars. There are three SNPs identified in the MYB28 allele: two located in the intronic region upstream of exon 3 and one at the 5’-end of exon 3 [120]. Sotelo et al. [121] used a DH mapping population of F1 from a Chinese kale and a DH broccoli line to detect the significant QTLs in leaves, flower buds, and seeds. They identified three loci controlling the content of aliphatic glucosinolates and four loci manipulating the accumulation of indole glucosinolates. Therefore, besides MYB28 allele from Brassica villosa, there might be other gene/locus variation determining the variety of aliphatic glucosinolate level in broccoli. In addition, significant general combining ability was observed between glucoraphanin concentration and total head content in 2-year assessment of 36 combinations by crossing nine pure parents’ lines [122]. Based on putative high-glucoraphanin materials, Gu et al. [86] also obtained sixteen high-glucoraphanin hybrids with acceptable agronomic traits by direct crossbreeding and screened two high-glucoraphanin parental lines.

The basic aims of breeding broccoli varieties with optimal glucosinolate composition and content include the following: (1) to reduce the content of anti-nutritional component, notably 2-hydroxy-3-butenyl glucosinolate; (2) to improve the content of beneficial compositions (glucoraphanin, glucoiberin, and glucoerucin) for better nutritional qualtiy, and to regulate the biosynthetic and hydrolytic pathways of glucosinolate profiles involved in interaction with pathegens and pests for enhanced resistance; (3) to promote the formation of ITCs rather than nitriles; (4) to preserve acceptable agronomic traits for head and considerable seed yield for sprout; and (5) to preserve other beneficial compounds, such as vitamins, minerals, and flavonoids. The related principles/strategies to reach above aims are as follows:

  1. 1.

    To select the parents without alkenyl glucosinolates through direct detection or markers (e.g., BoGS-ALK and BoGS-OH) selection.

  2. 2.

    To detect glucosinolates directly or analyze the critical genes or markers, which could regulate the aliphatic glucosinolate accumulation in edible parts. For example, the microsatellite marker OI12-F02 or other primers designed for the BoMYB28 allele, which are located in chromosome 2.

  3. 3.

    Due to no available markers for breakdown products variation, direct detection of breakdown products is required and enough for determination of the ability to deliver aliphatic glucosinolates to correspondent ITCs.

  4. 4.

    Other nutrients are not affected in the final hybrid. Furthermore, glucosinolate metabolism is widely involved in various defense reactions, especially against insects and pathogens. It is needed to consider the adaptability and resistance of glucosinolate hybrid in complex environments.

In addition to genotype, glucosinolate contents in broccoli are also influenced by many factors along the whole food chain from farm to table. Preharvest factors are pivotal to enhancement of glucosinolate accumulation, and suitable post-harvest handlings are essential for maintaining the glucosinolate content of broccoli products during the storage after harvest, while processing and cooking are other effective ways for glucosinolate retention before consumption.

5 Factors Influencing Glucosinolate Accumulation in Broccoli

5.1 Preharvest Factors

Plenty of investigations have been conducted to search useful tools for modulation of glucosinolate accumulation in broccoli before harvest. Regulation of glucosinolate by light as an environmental factor, as well as by phytohormones, sugars, salinity, and fertilization as chemical regulation , has been widely studied, and many valuable results were obtained.

5.1.1 Light

Light is a crucial environmental factor in plant life, which regulates seed germination, phototropism, and flowering [123, 124]. Recent studies have revealed that light influenced the accumulation of phytochemicals such as vitamin C, phenolic compounds and carotenoids, as well as glucosinolates [125–128]. It has been reported that UV-B radiation caused an induction of glucosinolate accumulation, especially of glucoraphanin and 4-methoxy glucobrassicin in broccoli sprouts and florets [129, 130]. In contrast, preharvest supplemental far-red light resulted in reduction of glucosinolate content, whereas supplemental red and blue light did not show a significant effect in broccoli florets [131].

5.1.2 Phytohormones

According to previous studies, glucosinolate accumulation in broccoli can be manipulated through treatment with several kinds of plant hormones, such as jasmonates , auxin, and brassinosteroids (BRs).

It has been shown that jasmonic acid (JA) and methyl jasmonate (MeJA) enhanced glucosinolate accumulation in broccoli. Liuann et al. [132] reported that significantly increased levels of indole glucosinolates including glucobrassicin, neoglucobrassicin, and gluconasturtiin were observed in 250 μM MeJA-treated broccoli (B. oleracea var. Green Magic), while the contents of aliphatic glucosinolates remained unchanged. Similar results were also observed in another individual survey, which demonstrated that the content of indole glucosinolates was increased by 3- to 20-fold upon JA treatment when compared to the control [133].

Since the indole acetaldoxime serves as a common precursor for indole glucosinolate and indole-3-acid (IAA) biosynthesis, close attention has been paid to the cross talk between these two kinds of indole compounds [134]. In broccoli, exogenous auxin treatment generally resulted in substantially higher levels of glucosinolates, especially indole glucosinolate. However, the effects of auxin varied with the types and concentrations used. IAA application at low concentration (0.1 mg L−1) led to the highest content of glucosinolates, followed by 0.1 mg L−1 indole-3-butyric acid (IBA), while 1 mg L−1 naphthalene acetic acid (NAA) treatment resulted in the lowest accumulation of glucosinolates [135].

Our previous survey has found that BR treatment downregulates glucosinolate accumulation in Arabidopsis [136], but a dose effect of BR existed in broccoli. Our other survey showed that the content of total glucosinolates and glucoraphanin in broccoli sprouts treated with 2 nM epibrassinolide (EBR) plus 40 mmol/L NaCl was increased by 86% and 85%, respectively. However, the reduction in glucosinolate content was observed due to the enhanced activity of myrosinase upon 20 nM EBR treatment alone [137].

5.1.3 Sugars

Sugar is not only an important source of carbon and energy but also an effective signal molecule modulating many developmental and metabolic processes in all phases of plant life cycle [138–143]. Our former studies have shown that glucosinolate accumulation in broccoli sprouts was enhanced by various kinds of sugars including sucrose, glucose, fructose, and mannitol [144, 145], with sucrose being the most effective one. Though mannitol treatment also led to an increase in glucosinolate content, the effect was not as strong as that resulted from sucrose treatment, which suggested that sucrose might function as a signal instead of osmotic stress in inducing glucosinolate accumulation [145]. Moreover, different concentrations of sugars have been demonstrated to have distinct influences on glucosinolate accumulation in broccoli sprouts. A relatively higher concentration of 176 mM sucrose and mannitol treatments dramatically increased glucosinolate content, whereas no significant difference was found after 88 mM sucrose or mannitol treatments [145]. As the glucosinolate level in broccoli is a reflection of two opposing physiological processes, glucosinolate biosynthesis and hydrolysis by myrosinase [146], two reasons may account for increased glucosinolates by sugar treatment. For one thing, the biosynthesis of glucosinolate might be induced by sugar treatment. It has been reported that Bo-ELONG, an important biosynthetic gene of aliphatic glucosinolate biosynthesis, was upregulated in broccoli sprouts by sucrose application [144]. For another, the activity of myrosinase was not changed after sucrose treatment, indicating that the hydrolysis of glucosinolate might not be affected by sugar treatment [144].

5.1.4 Salinity

Plants exhibit morphological or physiological alterations when subjected to salt stress. Besides, several reports indicated that salt stress was an important abiotic factor regulating glucosinolate accumulation in broccoli. The research by Carmen et al. [147] demonstrated that the total glucosinolate content in broccoli leaves was significantly increased after salt treatment and application of 80 mM NaCl displayed a more obvious effect when compared with 40 mM NaCl. This result was consistent with the data obtained from another research, which indicated a tendency of increase for the total glucosinolates in broccoli leaves upon NaCl treatment at concentrations of 60 mM and 90 mM [148]. However, studies in broccoli sprouts presented a contradictory result. We found that application of 40 mM and 80 mM NaCl did not notably enhance the accumulation of glucoraphanin in 4-day-old broccoli sprouts [149]. Similarly, our another study based on 7-day-old broccoli sprouts showed that total glucosinolate level was markedly decreased after treatment with NaCl at concentrations of 20, 40, and 60 mM [150]. These different responses might be due to rapid growth of sprouts in which Na+ and C1− ions become utilizable or different broccoli organs used for analysis [150].

5.1.5 Fertilization

The content of glucosinolate, a category of nitrogen- and sulfur- containing secondary metabolites, could be remarkably affected by nitrogen (N) and sulfur (S) fertilization. It has been demonstrated that levels of glucosinolates in broccoli were decreased by N fertilization but increased by N deficiency [151–153]. Schonhof et al. [152] reported that the elevation of glucosinolate content by N deficiency was mainly attributed to the presence of the alkyl glucosinolates, glucoraphanin and glucoiberin. However, in other Brassica crops, such as oilseed rape and Indian mustard, the levels of glucosinolates increased upon N application [154, 155]. This distinct response might be due to the difference of species used. In contrast to N fertilization, S fertilization usually led to an increase of glucosinolate content in broccoli in many cases [156]. However, conflicting result has also been demonstrated by another report which showed that broccoli sprouts did not benefit from S fertilization [157].

In addition to N and S fertilization, other fertilizer applications have also been indicated to influence glucosinolate accumulation. It has been reported that total glucosinolate contents in two broccoli cultivars (‘Calabrese’ and ‘Southern star’) were notably increased by organic and bioorganic fertilizers supply, respectively [158]. This is probably because organic and bioorganic manure can serve as alternative mineral fertilizers to improve soil structure [159] and microbial biomass [160].

Selenium (Se), an essential trace element for humans and mammals, often substitutes for S in physiological and metabolic processes in plants due to their chemical and physical resemblance [161]. Selenite and selenate salts, which are taken from the soil by plants through the sulfate absorption pathway, have been shown to improve the antioxidant status of plants. In addition, selenoglucosinolates displayed a higher anticarcinogenic activity than thioglucosinolates [162]. Se fertilization has been reported to exert no significant influence on glucosinolate levels in both broccoli sprouts [163–165] and florets [166]. Hence, Se fertilization is a good approach to accumulating selenium while maintaining glucosinolate content.

5.2 Post-Harvest Handlings

Broccoli is a highly perishable product, whose shelf life and visual quality strongly rely on storag e conditions, such as temperature, atmosphere composition, and relative humidity (RH) [146, 167, 168]. Generally, the declines of their visual quality are accompanied by the loss of nutrients including glucosinolate. Cooling, controlled atmosphere (CA), and modified atmosphere packaging (MAP) are widely used methods to extend the shelf life and reduce the nutrient loss. In addition, inhibiting the action of ethylene by 1-methylcyclopropene (1-MCP) treatment is also an effective way to improve the shelf life and quality of horticultural crops. The effects of these post-harvest handlings on glucosinolates of broccoli are discussed here.

5.2.1 Temperature

Glucosinolate contents generally decrease during post-harvest storage, which is coincided with the loss in visual quality of broccoli, and low temperatures clearly delay the quality decline [167]. Rangkadilok et al. [169] reported a more than 50% decrease in glucoraphanin concentration in ‘Marathon’ broccoli heads after 7-day storage at 20 °C. In contrast, no considerable decline was found after 7-day storage at 4 °C [169]. These results were consistent with the finding of Rodrigues and Rosa [170] that glucoraphanin levels declined to 82% when principal inflorescence of broccoli was left at 20 °C for 5 days but only 31% at 4 °C. The possible explanation is that plant cells are most likely to rapidly become damaged due to the loss of cellular integrity under high temperature and thus allowed the mixing of myrosinase and glucosinolates, resulting in the degradation and rapid decrease of glucosinolates. However, contradictory results were found in several other surveys. The study of Howard et al. [171] showed that sulforaphane decreased by approximately 50% after 21-day storage at 4 °C, and the severe decrease occurred within the first 7 days after harvest. Interestingly, the content of 4-methoxy glucobrassicin in broccoli florets stored at 10 °C increased from 0.4 μmol · g−1 DW to 1.8 μmol · g−1 DW after 9-day storage [172]. Likewise, the contents of 4-hydroxy glucobrassicin and 4-methoxy glucobrassicin in broccoli increased notably after chopping and 48 h storage at room temperature [173]. Moreover, the elevated level of 4-methoxy glucobrassicin in broccoli florets during the first day of storage at 20 °C was also observed by Yuan et al. [174]. It seems that the post-harvest enhancement of some indole glucosinolates counteracted the myrosinase-mediated degradation. Verkerk et al. [173] also proposed a stress-induced increase of glucosinolates by yet unknown mechanism, which plays a vital role in maintaining glucosinolate content of broccoli during post-harvest storage.

In addition to refrigeration, freezing is widely used in food industry. Usually, products are blanched before freezing, which can inactivate myrosinase as well as other enzymes causing deterioration. Rodrigues and Rosa [170] regarded freezing as the best method for preserving the glucosinolates in broccoli. However, blanching before freezing led to severe loss of glucosinolates by leaching them into water [175, 176]. Considering this, refrigeration might be the better procedure for storage of broccoli in comparison with freezing.

Although “cool as soon as possible” is the general recommendation for broccoli handling, delays of several hours before cooling may be encountered in post-harvest handling, especially when forced air or hydrocooling is employed instead of immediate liquid ice cooling in the field. Nevertheless, our former study showed that keeping broccoli florets at 20 °C for 6 h before cooling at 5 °C resulted in little influence on glucoraphanin content as well as shelf life, while keeping them at 20 °C for 24 h led to remarkable decline [168].

5.2.2 Controlled Atmosphere Storage

CA has been widely used to extend the storage period and maintain the quality of horticultural products. Broccoli is one of the commodities that benefit from CA storage [169, 177]. In regard to the changes of glucosinolate contents in broccoli upon CA treatment, discrepant results were obtained in various broccoli cultivars under different storage temperatures combined with diverse O2 and CO2 concentrations. Hansen et al. [172] investigated the changes of glucosinolates in ‘Marathon’ broccoli florets stored under low O2 and high CO2. Results showed that the total glucosinolate content increased 42% under air and 21% under 0.5% O2 + 20% CO2 during 7-day storage at 10 °C when compared to freshly harvested broccoli. However, the study of Fernández-León et al. [178] demonstrated that CA storage (10% O2, 5% CO2) was effective in lowering the decrease of glucosinolates in broccoli heads during cooling as well as room temperature (20 C) storage. Similarly, glucoraphanin concentration in broccoli heads was maintained to a higher level under CA (1.5% O2 + 6% CO2) storage at 4 °C than that under air condition [169]. These were consistent with the results of our former survey, which showed that CA conditions with relative higher CO2 concentrations (10%) and normal O2 concentrations (21%) facilitated the retention of glucoraphanin content at 5 °C [168]. These results suggested that the elevated CO2 concentration might favor the induction of glucoraphanin biosynthesis and/or reduction of its degradation by endogenous myrosinases. Dunford and Temelli [179] have proposed that elevated CO2 concentrations caused myrosinase inactivation, which might explain the decreased glucoraphanin degradation in CA conditions with elevated CO2 concentrations. Contrarily, marked decline in glucoraphanin content was found under 1% O2 or 1% O2 + 10% CO2 treatments compared to air treatment during 20-day storage at 5 °C [168], and 20% CO2 treatment led to a 15% decline of total glucosinolate content in ‘Marathon’ florets during 7-day storage at 10 °C [172]. It is known that CYP79F1, a cytochrome P450 monooxygenase , is responsible for aldoxime production, a key step in biosynthesis of glucoraphanin [16, 30, 180]. Therefore, the oxygen dependence of CYP79F1 action might explain above findings that low concentrations or the absence of O2 might result in reduced biosynthesis and content of glucoraphanin. Moreover, treatment with 20% CO2 in the absence of O2 brought about severe off-odors, injury, and water soaking of the tissue [172]. Thus, normal level of O2 and enhanced level of CO2 are better for CA treatment of broccoli.

5.2.3 Modified Atmosphere Packaging

Cooling and CA are effective in storage of broccoli [181]. However, cooling and CA facilities are not always available in developing countries, where high temperatures are often encountered throughout the post-harvest storage, distribution, and marketing phases of broccoli products [167]. In this case, MAP might be a better choice, which is simple, economical, and also effective in delaying the post-harvest deterioration and maintaining the visual quality of broccoli at either low or high temperature [146, 167, 169, 182]. The investigation of Rangkadilok et al. [169] showed that glucoraphanin content in broccoli heads descended significantly in air packaging at 20 °C with 48% loss at day 3 and 64% loss at day 10, whereas there were no significant changes in glucoraphanin levels in MAP with no holes at 4 °C and two microholes at 20 °C for up to 10 days. In this research, products were wrapped using low-density polyethylene (LDPE) bags (40 μm thick), and the atmospheres of approximately 0% O2 and 21% CO2 were reached in the MAP with microholes after 10 days at 20 °C, while the atmospheres inside the MAP (with no holes) were modified to 0.2% O2 and 15% CO2 after 10 days at 4 °C [169]. However, the glucoraphanin content dropped 48% in ‘Marathon’ heads after 7-day storage at 1 °C under MAP using 11 μm LDPE bags, in which the atmospheres reached 17% O2 and 2% CO2 [183].

A survey by Schreiner et al. [184] demonstrated that modified atmosphere, 8% O2 and 14% CO2, could maintain aliphatic and indole glucosinolates in mini broccoli heads for 7 days after an initial decrease in 4 days in mixed packaging of mini broccoli and mini cauliflower in polypropylene food trays. Similarly, the decreases of aliphatic, indole, and aromatic glucosinolates in ‘Parthenon’ broccoli florets were more severe when stored in air than that in modified atmospheres under microperforated polypropylene plastic after 12 days at 5 °C [182]. We also investigated the effects of MAP treatments on glucosinolate contents in ‘Youxiu’ broccoli heads by comparing with non-wrapped florets [146]. The broccoli florets were packaged by using polyethylene bags (40 μm thick) with no holes (M0), two microholes (M1), and four macroholes (M2) and then stored at 4 °C or 20 °C. The results showed that all three MAP treatments slowed the decline of glucosinolates in broccoli florets when compared to those in the control, with M0 being the most significant, followed by M1 and M2 during 23-day storage at 4 °C or 5-day storage at 20 °C.

In conclusion, MAP has potential in maintaining glucosinolates in broccoli at both low and high temperatures, while the differences in types of polyethylene film used, atmosphere reached in MAP, and genotypes of broccoli had distinct effects in preserving glucosinolates. In addition, it was worth noting that not only atmosphere but also RH was modulated by CA and MAP. Glucosinolate retention could benefit from these two factors as they could prevent cell membrane degradation and subsequent mixing of glucosinolates with myrosinase [167], though the precise mechanism involved remains to be further investigated.

5.2.4 1-Methylcyclopropene

1-MCP has been commercialized and widely utilized in post-harvest handling of fruits and vegetables. Ku et al. [185] indicated that application of 1-MCP could increase the storage life of broccoli. 1-MCP has also been found to maintain phytochemicals including glucosinolates in broccoli. We found that 1-MCP treatment at the concentration of 2.5 μl l−1 reduced the decreasing rate of total glucosinolates in broccoli florets stored at 20 °C [174]. Similar result was also observed by Fernández-León et al. [178] in broccoli heads under 0.6 μl l−1 1-MCP treatment. Moreover, the content of total glucosinolate was remarkably enhanced by application of 1-MCP (25 μl l−1) in broccoli florets stored at 15 °C for 5 days [186]. We found that 1-MCP treatment could inhibit the increase of malondialdehyde (MDA) when compared with the control [174]. It is known that MDA is the product of membrane peroxidation, which could damage the structure and integrity of membrane during the senescence of broccoli florets. Thus, lower MDA was beneficial for preventing the mixing of glucosinolates with myrosinase, namely, reducing glucosinolates hydrolysis. Moreover, the biosynthesis of glucosinolate is proved to participate in plant defensive response, and ethylene might play a role in this process [187]. Finally, it is also possible that the blockage of ethylene action by 1-MCP treatment favored glucosinolate biosynthesis or inhibited some ethylene-related degradative pathways. However, more studies are needed to elucidate the regulation mechanism of 1-MCP in glucosinolate metabolism.

In summary, application of 1-MCP is effective in delaying the decrease in glucosinolate contents during storage at low or relatively high temperature. Furthermore, 1-MCP has been proved nontoxic for humans and environment [188], and it is active at very low concentrations with a negligible residue. With no doubt, commercialization of 1-MCP will be a new tool to maintain glucosinolates in broccoli during post-harvest storage.

In addition, light and 6-benzylaminopurine (6-BA) treatments were also used in glucosinolate retention in post-harvest broccoli. Jin et al. [189] reported that the content of total glucosinolates in post-harvest broccoli florets was profoundly boosted by light-emitting diode (LED) green light . The levels of glucosinolate and sulforaphane in broccoli florets were markedly increased after treatment with 6-BA [190]. It is known that LED lighting sources are durable with small size and cool emitting surfaces, which is suitable for plant growth. 6-BA has been considered nontoxic for human and environment by the US Environment Protection Agency (EPA). Therefore, their applications in post-harvest handling of broccoli are potential.

5.3 Processing and Cooking

Broccoli is mostly consumed as a processed food, and various processings before consumption might cause the degradation or transformation of the health-promoting compounds including glucosinolates. Conventional processing and cooking methods generally affect the content of glucosinolate in broccoli by several aspects: (I) glucosinolate leakage into cooking water, (II) enzymatic hydrolysis by myrosinase, and (III) glucosinolate breakdown in thermal conditions [191]. Here, we try to summarize the effects of processing and cooking conditions on glucosinolate and its derivative in broccoli.

5.3.1 Blanch-Freezing

Broccoli is almost exclusively available to the consumer in two forms, fresh broccoli heads and frozen broccoli florets. Freezing is widely used in broccoli processing as it provides cheaper product with longer shelf life. In commercial industrial freezing , broccoli usually undergoes blanching prior to freezing, a processing method utilizing hot water or steam to inactivate enzymes that may cause degradative changes and thus limit shelf life severely [192]. Unfortunately, blanching also destroyed myrosinase, resulting in the disability to form sulforaphane in pre- and post-cooking in frozen broccoli, and substantially reduced sulforaphane bioavailability [193]. Because of the disnatured myrosinase, blanch-frozen broccoli can retain the same levels of glucosinolates after 90 days of frozen (−20 °C) storage [194]. Furthermore, Alanís-Garza et al. [195] reported that glucosinolate content increased in three tested broccoli cultivars while remained constant in only one cultivar after blanching and freezing. In order to maximize the production of cancer preventative sulforaphane in broccoli florets, blanching step is optimized to maintain myrosinase activity while destroying ESP activity, for a high percentage of glucoraphanin can be converted to a nitrile with ESP as mentioned above. Perez et al. [196] proposed that the optimal blanching conditions to maximize sulforaphane content in broccoli florets were immersion in water at 57 °C for 13 min, coinciding with the minimum glucosinolates and maximum myrosinase activity.

5.3.2 Cooking

Broccoli is always cooked before eating. Domestic cooking methods include boiling, steaming, microwaving, stir-frying, and stir-frying followed by boiling (stir-frying/boiling). All these methods could influence the levels of glucosinolates and their hydrolysis products in broccoli and thus affect its heath protective capacity. Slicing is the common step in food preparation prior to further cooking, which disrupted tissues and facilitated the release of myrosinase, leading to glucosinolate hydrolysis at high degree [146]. General heating methods in cooking such as boiling, microwaving, and steaming would cause decrease of glucosinolate content in broccoli, and the rate of decrease was higher along with increased cooking time [197]. Sones et al. [198] found that the total glucosinolate content of broccoli boiled for 10 min was approximately 40% less than that of fresh broccoli. Moreover, the total glucosinolate content dropped by 62.0% and 67.7% after 5 and 10 min of blanching, respectively, with the greatest decline in glucoraphanin (71.58%) being observed after 1 min of boiling [197]. Boiling with cold start and hot start also shows different glucosinolate retention. A glucosinolate decrease of 50% was observed in boiling-cold start while 41% loss in boiling-hot start, which provide a suggestion for consumers in preferring boiling way [199]. The microwave heating process led to distinct results due to the differences in conditions such as cooking time, power, and the volume of added water [200]. Vallejo et al. [201] reported a reduction of 74% in total glucosinolate concentration after microwaving broccoli at 1000 W for 5 min, which is the result of glucosinolate leaching into the evaporated water. Likewise, a significant loss of glucosinolates (62% reduction in glucoraphanin) was also observed in microwaved broccoli in our former study [202]. As for steaming, our survey showed that it led to a 36.8% loss of total indole glucosinolates and no significant change in the content of total aliphatic glucosinolates [202]. Similarly, steaming caused a decrease of 17% in total glucosinolate content after cooking for 22 min [199]. Interestingly, Gliszczynska-Swiglo et al. [203] found that steaming elevated glucosinolate content in broccoli when compared with the fresh broccoli. Bongoni et al. [199] also reported that steaming increased total glucosinolates’ level by 17% at the end of cooking, which may be attributed to an increase in the extractability of glucosinolates by processing rather than a real increase in their total content. In addition, the contents of phytochemicals including glucosinolate were evaluated when fresh broccoli florets were subjected to stir-frying treatments in various edible oils, and the results indicated that glucosinolate in broccoli stir-fried with extra-virgin olive, soybean, peanut, or sunflower oil was similar to that in the uncooked sample [204]. We compared the effect of all five domestic cooking methods on the retention of glucosinolates in broccoli and found that stir-frying and stir-frying/boiling presented the highest loss of glucosinolates while steaming resulted in the lowest loss [202]. Therefore, steaming method appeared to be the best way to retain glucosinolates in cooking broccoli.

The loss of glucosinolate in broccoli upon cooking is mainly caused by leaching, for numerous glucosinolates were found in cooking media [205, 206]. Furthermore, heating also leads to thermal degradation of glucosinolate. In general, aliphatic glucosinolates are more stable than indole glucosinolates during cooking [197, 201, 202]. The relative thermostability of individual glucosinolates was shown to vary with heating temperature, duo to their respective chemical structures [207, 208]. However, thermal degradation might not be the main cause of glucosinolate loss as the content of glucosinolates dropped considerably within a very short cooking time.

In addition, significant change in sulforaphane content was detected in cooked broccoli. Matusheski et al. [209] showed that sulforaphane production increased when broccoli was cooked at 60 °C for 5 or 10 min, as temperature s higher than 50 °C inactivated ESP. Considering that myrosinase could be inactivated when subjected to 100 °C for 5–15 min, thus cooking at temperature between 50 °C and 90 °C or at 100 °C within 5 min would favor the formation of sulforaphane [209]. This conclusion was also supported by another survey, which found that steaming for 1–3 min produced less nitrile and more sulforaphane yield from a broccoli meal [65]. It has also been reported that microwave heating (900 W) for 0.5 and 0.75 min increased sulforaphane in production while reduced sulforaphane nitrile content. Furthermore, Ghawi et al. [210] found that adding mustard seeds was proved to intensify sulforaphane formation in cooked broccoli, as these seeds contained a more resilient isoform of myrosinase.

5.3.3 Other Processing Methods

An early publication by Rosa et al. [211] reported the effect of dehydration processes on glucosinolates in broccoli. They found that 50–65 °C drying of intact broccoli maintained the original glucosinolate content as well as the myrosinase activity. However, the rehydration process caused the hydrolysis of glucosinolates. Moreover, as ESP is disnatured during drying (50–65 °C), sulforaphane synthesis should be favored in dehydration processes. A recent study suggested that pulsed electric field (PEF) treatment could increase glucosinolate content in both floret and stalk of broccoli, and the optimal condition was 4 kV cm−1 for 525 and 1000 μs [212]. Other processing conditions, such as pH, also had a significant effect on sulforaphane and sulforaphane nitrile production. A neutral or alkaline pH resulted in predominate sulforaphane production, whereas an acidic pH (3.5, typical of salad dressings) led to more sulforaphane nitrile [108].

6 Conclusions

In recent years, a great progress has been achieved in elucidating glucosinolate biosynthetic pathway, as well as its regulation and degradation in broccoli by a combination of molecular, genomic, and bioinformatic approaches, which provides a solid basis for breeding of broccoli for optimal glucosinolate composition and content. However, plant secondary metabolites usually have adverse effects besides beneficial properties, and several glucosinolate breakdown products have anti-nutritional effects. Therefore, more studies are needed to identify the biological activities of the glucosinolate degradation products in greater detail, so that the balance of benefit, risk, and consumer preference can be properly defined.

Currently, metabolic engineering of glucosinolate in Brassica crops is underway, providing a feasible method for improving glucosinolate composition and content in these crops [213–217]. A thorough understanding of glucosinolate biosynthetic pathway and its regulatory network is necessary for regulation of glucosinolate in broccoli by molecular breeding and metabolic engineering, which is promising for better nutrition value and resistance.

In addition, a large number of preharvest factors and post-harvest handlings have been clarified to modulate glucosinolate accumulation in broccoli, and attentions are paid to glucosinolate metabolism in broccoli across the whole food chain, from the production to consumption. Suitable environmental conditions such as light, as well as chemical regulations, help to enhance glucosinolate accumulation during production before harvest. Post-harvest handlings including cooling, CA, MAP, and 1-MCP treatments, as well as freezing processing, are effective in attenuating glucosinolate loss during post-harvest package, storage, and processing period, and steaming is the recommended method for best retention of glucosinolate during cooking before consumption in broccoli. However, the underlying mechanism behind regulation of glucosinolate metabolism by different preharvest and post-harvest handlings remains to be further elucidated.