Sulforaphane Formation Research Articles

Lactobacillus plantarum increase the sulforaphane formation efficiency via microbial-targeted delivery system in vivo

Sulforaphane is produced by glucoraphanin under the catalysis of myrosinase in crushed cruciferous plants and is well known for its anti-inflammatory, bacteriostatic and functions. Researches have shown that microorganisms could convert glucoraphanin into sulforaphane in human body and natural environment. However, the transformation efficiency of sulforaphane by intestinal microorganisms is not sufficient. In order to improve human health, this research focused on screening and delivering probiotics with high sulforaphane transformation efficiency into human body and promoting probiotics to produce more sulforaphane. In this research, microorganisms capable of transforming sulforaphane in nature were screened, identified, and further delivered to intestine through a microbe-targeted delivery system (thiolated oxidized konjac glucomannan, sOKGM) to improve the in vivo transformation efficiency of sulforaphane. The results showed that the sulforaphane was converted from glucoraphanin with higher transformation rate of 0.1863 × 10−4 mol h−1 logCFU−1 in Lactobacillus plantarum than that of L. acidophilus and L. paracasei. In addition, the encapsulation efficiency of L. plantarum by microbe-targeted delivery system was 87.02%, with the survival rate of 28.33%. The sulforaphane content of embedded L. plantarum group was 3.075 × 10−3 mol L−1, which was significantly higher than that of not embedded L. plantarum group (1.430 × 10−3 mol L−1) after digestion with simulated gastrointestinal fluid. This research revealed that L. plantarum could effectively improve the transformation efficiency of sulforaphane in human intestines, providing strong experimental evidence to support the high-value utilization of sulforaphane and enhancement of human health.

Impact of Different Technologies and Methods to Increase the Shelf Life and Maintain the Sulforaphane Content in Broccoli: A Review

Broccoli, a nutrient-rich vegetable known for its sulforaphane content and health benefits, faces challenges related to its short shelf life and susceptibility to spoilage. This study explores various techniques to maintain sulforaphane content and extend broccoli's shelf life. Freezing, although effective for long-term preservation, involves blanching that inactivates myrosinase, preventing sulforaphane formation. Cold storage at 4-5°C slows respiration and senescence, extending shelf life up to 14 days while preserving bioactive compounds. Modified atmosphere packaging (MAP) enhances shelf life by altering the package atmosphere, with active packaging using 15% CO2 and 7.5% O2 at 0°C proving particularly effective in reducing bacterial growth and preserving quality. Light exposure, especially with yellow LED lights and moderate UV treatment, retains high levels of glucosinolates and phenolic compounds. Irradiation effectively eliminates pathogens and preserves visual quality, with gamma irradiation slowing the oxidation of phenols and ascorbic acid, although further research is needed on its sensory and nutritional effects. High-pressure processing (HPP), a non-thermal method, significantly boosts sulforaphane bioactivity by converting glucosinolates at pressures up to 600 MPa, despite challenges such as high costs and operational safety, and prevents microbial growth, extending shelf life while maintaining nutritional quality. Additionally, techniques like steaming for optimal durations, microwaving at high power for short times, and stir-frying help preserve sulforaphane content during cooking. Preharvest chemical treatments like chitosan and calcium also enhance broccoli's defence mechanisms and bioactive compound levels. Overall, these methods offer a comprehensive approach to preserving sulforaphane content and extending broccoli's shelf life, providing valuable insights to enhance the storage, quality, and health benefits of broccoli for consumers.

Application of Tryptophan and Methionine in Broccoli Seedlings Enhances Formation of Anticancer Compounds Sulforaphane and Indole-3-Carbinol and Promotes Growth.

Broccoli is a popular cruciferous vegetable that is well known for its abundant health-promoting biochemicals. The most important of these beneficial biochemicals are glucosinolates, including glucoraphanin and glucobrassicin. Glucoraphanin and glucobrassicin can be broken down by myrosinases into sulforaphane and indole-3-carbinol, which have been demonstrated to have potent cancer-preventive properties. Efforts to increase glucoraphanin in broccoli seedlings have long been a focus; however, increasing glucoraphanin and glucobrassicin simultaneously, as well as enhancing myrosinase activity to release more sulforaphane and indole-3-carbinol, have yet to be investigated. This study aims to investigate the impact of the combined application of tryptophan and methionine on the accumulation of sulforaphane and indole-3-carbinol, as well as their precursors. Furthermore, we also examined whether this application has any effects on seedling growth and the presence of other beneficial compounds. We found that the application of methionine and tryptophan not only increased the glucoraphanin content by 2.37 times and the glucobrassicin content by 3.01 times, but that it also caused a higher myrosinase activity, resulting in a1.99 times increase in sulforaphane and a 3.05 times increase in indole-3-carbinol. In addition, better plant growth and an increase in amino acids and flavonoids were observed in broccoli seedlings with this application. In conclusion, the simultaneous application of tryptophan and methionine to broccoli seedlings can effectively enhance their health-promoting value and growth. Our study provides a cost-effective and multi-benefit strategy for improving the health value and yield of broccoli seedlings, benefiting both consumers and farmers.

BoMyrosinase plays an essential role in sulforaphane accumulation in response to selenite treatment in broccoli

Sulforaphane, a naturally specialized metabolite, plays significant roles in human disease prevention and plant defense. Myrosinase (MY) is a key gene responsible for the catalysis of sulforaphane formation, but the molecular mechanisms through which MY regulates sulforaphane biosynthesis in plants remains largely unknown. Here, we discovered that the change of sulforaphane content in broccoli sprouts caused by exogenous selenite treatments is positively related to BoMY expression. BoMY overexpression in the Arabidopsis thaliana tgg1 mutants could dramatically increase myrosinase activity and sulforaphane content in the rosette leaves of 35S::BoMY/tgg1 and rescue its phenotypes. Moreover, an obvious increase of myrosinase activity and sulforaphane content was displayed in transgenic BoMY-overexpressed broccoli lines. In addition, a 2 033 bp promoter fragment of BoMY was isolated. Yeast one-hybrid (Y1H) library screening experiment uncovered that one bHLH transcription factor, BoFAMA, could directly bind to BoMY promoter to activate its expression, which was further evidenced by Y1H assay and dual-luciferase reporter assay. BoFAMA is a selenite-responsive transcription factor that is highly expressed in broccoli leaves; its protein is solely localized to nucleus. Additionally, genetic evidence suggested that the knockdown of FAMA gene in Arabidopsis thaliana could significantly decrease sulforaphane yield by inhibiting the expression of myrosinase genes. Interestingly, exogenous selenite supply could partially restore the low level of sulforaphane content in transgenic Arabidopsis FAMA-silencing plants. Our findings uncover a novel function of FAMA-MY module in the regulation of selenite-mediated sulforaphane synthesis and provide a new insights into the molecular mechanism by which selenite regulates the accumulation of sulforaphane in plants.

Enhancement of ultrasound-assisted extraction of sulforaphane from broccoli seeds via the application of microwave pretreatment

In this study, microwave pretreatment and grinding treatment were used to enhance sulforaphane formation, then ultrasonic-assisted extraction (UAE) was applied to extract sulforaphane using simultaneous hydrolysis and extraction method. The effects of various parameters, which were ultrasonic time,ultrasonic power, solid-water ratio and solid-ethyl acetate ratio on the extraction rate of sulforaphane were investigated. The results showed that microwave pretreatment enhanced sulforaphane formation. Excessive size reduction did not increase or even reduced extraction rate of sulforaphane. Simultaneous hydrolysis and extraction significantly increased extraction rate of sulforaphane compared to hydrolysis followed by extraction. UAE accelerated mass transfer and the solubilization of the targeted compounds due to the acoustic cavitation effect, thus enhanced enzymatic hydrolysis of glucoraphanin and the extraction rate of sulforaphane. The extraction rate of sulforaphane using UAE with simultaneous hydrolysis and extraction was 4.07-fold of the conventional extraction method. UAE was an effective method to extract sulforaphane from broccoli seeds since it led to higher yield of sulforaphane in a much shorter extraction time.

Effect of different LED lights on aliphatic glucosinolates metabolism and biochemical characteristics in broccoli sprouts

In this study, various LED lights (white, red, yellow, green, blue and purple) were applied to evaluate their effects on the biochemical parameters, aliphatic glucosinolates synthesis and sulforaphane formation in broccoli sprouts. The length of broccoli sprouts and bulk water content were decreased after LED light treatments, especially white, green, and purple LED lights. The highest contents of ascorbic acid and anthocyanins were recorded in blue LED light treated broccoli sprouts. Green LED light treated sprouts contained the highest contents of ATP, ADP and AMP. Yellow and bule lights resulted in higher glucose content. Yellow, blue, and purple LED lights induced glucoraphanin and glucoerucin accumulation via upregulating aliphatic glucosinolates biosynthetic genes. Yellow LED light caused higher glucoraphanin content than blue LED light, while sulforaphane formation showed no difference; which might be attributed to higher myrosinase activity and its expression as well as lower ESM1 expression under blue LED light.

Purple light-emitting diode (LED) lights controls chlorophyll degradation and enhances nutraceutical quality of postharvest broccoli florets

The aim of this study was to evaluate the effect of white, red, green, yellow, blue and purple LED lights on visual and nutraceutical quality of broccoli florets during storage at 20 °C for 5 days. The results showed that purple LED light exhibited the best effect on reducing yellowing and retaining chlorophyll via down-regulating expression of genes related to chlorophyll degradation, including BoSGR, BoPAO, BoNYC1 and BoRCCR. On the other hand, yellow, blue and purple LED light increased contents of total phenolic and carotenoids compared to darkness. The highest glucoraphanin content was recorded in purple LED light treated broccoli florets, which was supported by the enhanced expression levels of genes related to glucoraphanin biosynthesis. Meanwhile, purple LED light promoted myrosinase activity and its expression, and reduced ESP expression; thus enhanced sulforaphane formation. In conclusion, among all LED lights, purple LED light showed a significant positive effect considering all estimated indicators and would be useful to maintain and improve the quality of broccoli during storage at 20 °C.

Inactivation of Escherichia coli on broccoli sprouts via plasma activated water and its effects on quality attributes

In the present study, the efficacy of PAW with different activation time and PAW treatment time in decontamination of Escherichia coli (E. coli) on broccoli sprouts and the effect of PAW on the quality of broccoli sprouts were investigated. The results showed that PAW60 treatment for 15 min resulted in the highest reduction of E. coli on broccoli sprouts. The bactericidal effect was also confirmed by the changes of morphology, cell membrane permeability and protein changes of E. coli cells via analyses of the scanning electron microscope (SEM), the flow cytometer and circular dichrograph. Meanwhile, no significant difference was observed in color, cellular structure and total phenolic content between PAW treatment and control. Moreover, PAW treatment did not affect or even enhanced sulforaphane formation in broccoli sprouts. These data indicate that PAW washing can be used a promising sterilization method to assure the food safety and quality of brcoccoli sprouts.

Effects of White and Red Light Photoperiods on Physiology and Biochemistry as well as Glucoraphanin Metabololism of Broccoli Sprouts

In this study, the effects of white light and red light at different photoperiods (0, 4, 8, 12, 16 and 20 h·d−1) on growth, contents of soluble sugar, soluble protein, ascorbic acid, anthocyanins as well as glucoraphanin metabolism of broccoli sprouts were studied. The results showed that the growth of broccoli sprouts was inhibited by light treatment, but red light promoted the growth compared with white light. With the increasing of light photoperiods, the content of soluble sugar, soluble protein, ascorbic acid, glucoraphanin and total glucosinolates, myrosinase activity as well as sulforaphane formation firstly increased, then decreased. Compared with white light, red light significantly enhanced soluble sugar, soluble protein, ascorbic acid and glucoraphanin content in broccoli sprouts. However, higher anthocyanin content was observed in white light treated broccoli sprouts rather than red light treated broccoli sprouts. In addition, when photoperiod was less than 12 h, red light resulted in higher sulforaphane formation than white light (P<0.05). When photoperiod was more than 12 h, red light did not affect even decreased sulforaphane formation compared with white light treatment. In conclusion, red light treatment was beneficial to the enhancement of yield and nutritional quality of broccoli sprouts compared with white light, and 12 h·d−1 was the appropriate photoperiods.

Factors Influencing Sulforaphane Content in Broccoli Sprouts and Subsequent Sulforaphane Extraction

Broccoli sprouts contain 10–100 times higher levels of sulforaphane than mature plants, something that has been well known since 1997. Sulforaphane has a whole range of unique biological properties, and it is especially an inducer of phase 2 detoxication enzymes. Therefore, its use has been intensively studied in the field of health and nutrition. The formation of sulforaphane is controlled by the epithiospecifier protein, a myrosinase co-factor, which is temperature-specific. This paper studies the influence of temperature, heating time, the addition of myrosinase in the form of Raphanus sativus sprouts in constant ratio to broccoli sprouts, and other technological steps on the final sulforaphane content in broccoli sprout homogenates. These technological steps are very important for preserving sulforaphane in broccoli sprouts, but there are some limitations concerning the amount of sulforaphane. We focused, therefore, on the extraction process, using suitable β-cyclodextrin, hexane and ethanol, with the goal of increasing the amount of sulforaphane in the final extract, thus stabilizing it and reducing the required amount sulforaphane needed, e.g., as a dietary supplement.

Calcium affects glucoraphanin metabolism in broccoli sprouts under ZnSO4 stress

CaCl2, Ca2+ chelator (EGTA) and Ca2+ channel blocker (verapamil) were used to investigate mechanism of glucoraphanin metabolism in broccoli sprouts under ZnSO4 stress. CaCl2 treatment promoted sprout growth, reduced MDA (malonaldehyde) content and electrolyte leakage in sprouts under ZnSO4 stress. The highest MDA content and electrolyte leakage were obtained in ZnSO4 plus verapamil-treated sprouts. In addition, ZnSO4 plus CaCl2 treatment significantly enhanced glucoraphanin content and sulforaphane formation, while an opposite result was observed after ZnSO4 plus EGTA treatment; which were further supported by expression of glucoraphanin biosynthetic and hydrolytic genes as well as myrosinase (MYR) and epithiospecifier protein (ESP) activities. These results indicated that exogenous and endogenous calcium promoted glucoraphanin biosynthesis and the conversion rate of glucoraphanin into sulforaphane. Verapamil treatment also stimulated glucoraphanin biosynthesis, but exerted an adverse influence on sulforaphane formation from the hydrolysis of glucoraphanin because of much higher ESP expression and ESP activity than ZnSO4 treatment.

Melatonin treatment affects the glucoraphanin-sulforaphane system in postharvest fresh-cut broccoli (Brassica oleracea L.)

The effect of postharvest melatonin treatment on sulforaphane production of fresh-cut broccoli at 4℃ during storage was investigated in this study. Florets treated with 100 μM melatonin exhibited higher contents of total glucosinolates and sulforaphane. Glucoraphanin content was significantly increased after melatonin treatment, and which was explained by gene analysis. Expressions of glucoraphanin biosynthesis genes including Elong, CYP83A1, MYB28, UGT74B1 and FMOGS-OX1 were up-regulated while AOP2 was obviously decreased by melatonin treatment, leading to a higher glucoraphanin accumulation. In addition, application of melatonin enhanced the myrosinase activity and the expression level of MYO, benefiting the formation of sulforaphane. This study demonstrates that melatonin treatment positively affected the glucoraphanin-sulforaphane system in postharvest fresh-cut broccoli.

Enhanced production of sulforaphane by exogenous glucoraphanin hydrolysis catalyzed by myrosinase extracted from Chinese flowering cabbage (Brassica rapa var. parachinensis)

Sulforaphane formation via endogenous route is known to be less effective. Exogenous hydrolysis of the sulforaphane precursor is therefore of interest. Here, myrosinase activity was first determined to identify a suitable source of the enzyme from selected Brassica vegetables. Extracted enzyme was then evaluated for its thermal stability to establish a condition for extraction. Chinese flowering cabbage was selected as the source of myrosinase; suitable extraction condition was at 40 °C for 90 min. Enzyme extract was used to hydrolyze glucoraphanin standard into sulforaphane at 30 °C and pH 6. Exogenous hydrolysis reached the equilibrium with the reverse reaction after 30 min; sulforaphane concentration remained unchanged afterward. Molar fractional conversion of glucoraphanin into sulforaphane at 30-min hydrolysis was around 48%. In comparison with exogenous hydrolysis by myrosinase extracted from broccoli, which indeed exhibits higher activity than the enzyme extracted from Chinese flowering cabbage, no conversion of glucoraphanin into sulforaphane was unexpectedly observed.

Methyl jasmonate, salicylic acid and abscisic acid enhance the accumulation of glucosinolates and sulforaphane in radish (Raphanus sativus L.) taproot

Radish is an important root vegetable crop. The effects of environmental stresses on glucosinolate (GLS) contents were extensively investigated in Brassicaceae species. However, little is known about the accumulation of GLS and sulforaphane (SF) in hormone-treated radish taproot. In this study, the effects of methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) on the accumulation of GLS and SF in radish taproot were investigated. The GLS content increased considerably in response to MeJA treatment. Moreover, MeJA also up-regulated the expression level of several transcription factors and GLS biosynthesis related genes. The effects of SA on GLS content were less obvious, while ABA suppressed GLS biosynthesis. Additionally, the SF content increased under treatments with MeJA, SA and ABA. Furthermore, changes in myrosinase activity and glucoraphanin (GRA) content after the application of MeJA, SA and ABA were consistent with the changes in SF contents. The results indicated that the MeJA represents one of the most effective plant growth regulators (PGRs) for increasing the GLS content in radish taproot by up-regulating the expression levels of a few GLS biosynthetic related genes. These findings also implied that SF formation is correlated with myrosinase activity and GRA content in radish taproot.

Comparative proteomics analysis of whitetop (Lepidium draba L.) seedlings in response to exogenous glucose

In this research, a comparative proteomics approach was conducted to understand the physiological processes behind the sulforaphane formation in whitetop seedlings in response to exogenous glucose. Initially, 5-day-old whitetop seedlings were elicited by different concentrations (0, 166, 250, 277, 360 mM) of glucose for 72 h. According to the results, sulforaphane formation was influenced in a dose-dependent manner by glucose, and was maximized with the concentrations of 166 and 250 mM. Consequently, 2-dimensional gel electrophoresis was performed on the 166 mM glucose-elicited seedlings and it was shown that 25 protein spots were differentially expressed between glucose-elicited seedlings and control. Two hypothetical (were down-regulated) and 9 unique proteins (44% and 56% up- and down-regulated, respectively) were identified based on the Mass spectrometry analysis. According to the functional classification of the unique proteins, photosynthetic, chaperone, energy metabolism, signaling and sorting related proteins are marked in response to the glucose elicitation. This is the first report to successfully identify the Abscisic acid receptor PYR1-like and sorting nexin 1 isoform X1 by proteomics technique. In addition, the role of the sorting nexin 1 isoform X1 in the glucose-elicited whitetop seedling is reported for the first time.

Formation of Sulforaphane and Iberin Products from Thai Cabbage Fermented by Myrosinase-Positive Bacteria.

Myrosinase-positive bacteria from local fermented foods and beverages in Thailand with the capacity to metabolize glucosinolate and produce isothiocyanates (ITCs) were isolated and used as selected strains for Thai cabbage fermentation. Enterobacter xiangfangensis 4A-2A3.1 (EX) from fermented fish and Enterococcus casseliflavus SB2X2 (EC) from fermented cabbage were the two highest ITC producers among seventeen strains identified by 16S rRNA technique. EC and EX were used to ferment Thai cabbage (Brassica oleracea L. var. capitata) containing glucoiberin, glucoraphanin and 4-hydroxyglucobrassicin at 430.5, 615.1 and 108.5 µmol/100 g DW, respectively for 3 days at 25 °C. Different amounts of iberin nitrile, iberin, sulforaphane and indole 3-acetonitrile were produced by spontaneous, EX- and EC-induced cabbage fermentations, and significantly higher ITCs were detected (p < 0.01) with increased antioxidant activities. Iberin and sulforaphane production in EX-induced treatment peaked on day 2 at 117.4 and 294.1 µmol/100 g DW, respectively, significantly higher than iberin at 51.7 µmol/100 g DW but not significantly higher than sulforaphane at 242.6 µmol/100 g DW in EC-induced treatment at day 2. Maximum health-promoting benefits from this functional food can be obtained from consumption of a liquid portion of the fermented cabbage with higher ITC level along with a solid portion.

Intensifying sulforaphane formation in broccoli sprouts by using other cruciferous sprouts additions.

Sulforaphane is a significant chemopreventive compound which is the predominant glucosinolate in broccoli sprouts. However, the existence of the epithiospecifier protein could direct the hydrolysis of glucosinolates toward sulforaphane nitrile formation instead of sulforaphane. Therefore, the study aimed on improving the yielding of sulforaphane in broccoli sprouts with a new method of the united hydrolysis of cruciferous sprouts. According to the results, the addition of radish, rocket and rape sprouts to broccoli sprouts could promote the hydrolysis of the glucoraphanin to anticancer effective sulforaphane to 2.03, 2.32 and 1.95-fold, respectively, compared to single broccoli sprouts. Meanwhile, the formation of non-bioactive sulforaphane nitrile in these three groups decreased greatly. However, the addition of mustard sprouts had no positive effect. These observations could make a contribution to the potential chemoprotective effects of broccoli sprouts.

Microwave pretreatment enhances the formation of cabbage sulforaphane and its bioaccessibility as shown by a novel dynamic soft rat stomach model

Microwave (MW) pretreatment was used to increase the sulforaphane content in cabbages. In vitro digestion in rat stomach and shaking flask was then investigated to monitor the bioaccessible sulforaphane content in the pretreated cabbages. Microstructural changes of cabbages were observed and used to support the pretreatment and digestion results. MW treatment at 26.40 W/g for 15 s increased the sulforaphane content by 6.23 times compared with that in the fresh samples. Bioaccessible sulforaphane content after rat-stomach digestion increased 3.48–4.19 times compared with that in the pretreated samples before digestion. This increase was similar to that after shaking-flask digestion, probably because cabbage tissues are soft and can be easily digested even in a simplified batch in vitro digestion system. Nevertheless, sulforaphane concentration in the digested mixture during rat-stomach digestion became constant after 15 min, while that in the shaking flask reached the same concentration only after 75 min.

Calcium sulfate treatment enhances bioactive compounds and antioxidant capacity in broccoli sprouts during growth and storage

The effect of preharvest CaSO4 treatment on antioxidant enzyme activities, bioactive compounds and antioxidant capacity in broccoli sprouts during growth and storage was investigated in this study. Application of CaSO4 increased the biomass and reduced electrolyte leakage of broccoli sprouts. Higher antioxidant enzyme activities and antioxidant capacity in sprouts were obtained in CaSO4-treated sprouts during growth and storage. Total phenolic content in CaSO4-treated sprouts was lower than that in control ones during growth; but was higher than in control sprouts during storage. Additionally, the decrease of ascorbic acid was suppressed by CaSO4 treatment during storage. CaSO4 treatment also dramatically enhanced glucosinolate content, especially glucoraphanin, in broccoli sprouts during growth and prevented its loss during storage. This was further supported by the up-regulation of glucoraphanin biosynthesis-related genes. During storage, CaSO4-treated sprouts exhibited higher myrosinase activity and lower ESP activity, which resulted from higher expression of MYR and ESM1. Moreover, CaSO4 treatment led to higher sulforaphane formation in sprouts than control during growth and storage.

Sulforaphane formation and bioaccessibility are more affected by steaming time than meal composition during in vitro digestion of broccoli

Broccoli is a rich source of the glucosinolate glucoraphanin (GR). After hydrolysis of GR by the endogenous enzyme myrosinase, sulforaphane (SF) or sulforaphane nitrile (SFN) are produced, depending on environmental conditions. How the conversion of GR and bioaccessibility of released breakdown products are affected by steaming (raw, 1min, 2min and 3min steamed) and meal composition (protein or lipid addition) was studied with an in vitro digestion model (mouth, stomach, intestine, but not colonic digestion).The main formation of SF and SFN occurred during in vitro chewing. The contents of GR, SF and SFN did not change after further digestion, as the irreversible inactivated myrosinase under gastric conditions caused no further GR hydrolysis. SF concentrations were up to 10 times higher in raw and 1min steamed broccoli samples after digestion compared to longer-steamed broccoli. Protein or lipid addition had no influence on the formation and bioaccessibility of SF or SFN.