Computer-driven Evolution of Myrosinase from the Cabbage Aphid for Efficient Production of (R)-Sulforaphane.

Myrosinase (Myr) catalyzes the hydrolysis of glucosinolates, yielding biologically active metabolites. In this study, glucoraphanin (GRA) extracted from broccoli seeds was effectively hydrolyzed using a Myr-obtained cabbage aphid (Brevicoryne brassicae) (BbMyr) to produce (R)-sulforaphane (SFN). The gene encoding BbMyr was successfully heterologously expressed in Escherichia coli, resulting in the production of 1.6 g/L (R)-SFN, with a remarkable yield of 20.8 mg/gbroccoli seeds, achieved using recombination E. coli whole-cell catalysis under optimal conditions (pH 4.5, 45 °C). Subsequently, BbMyr underwent combinatorial simulation-driven mutagenesis, yielding a mutant, DE9 (N321D/Y426S), showing a remarkable 2.91-fold increase in the catalytic efficiency (kcat/KM) compared with the original enzyme. Molecular dynamics simulations demonstrated that the N321D mutation in loopA of mutant DE9 enhanced loopA stability by inducing favorable alterations in hydrogen bonds, while the Y426S mutation in loopB decreased spatial resistance. This research lays a foundation for the environmentally sustainable enzymatic (R)-SFN synthesis.

Accumulation of Sulforaphane and Alliin in Human Prostate Tissue.

Diets rich in cruciferous vegetables have been associated with a lower risk of incidence and progression of prostate cancer. Sulforaphane, an isothiocyanate derived from 4-methylsulphinylbutyl glucosinolate (glucoraphanin) that accumulates in certain of these vegetables, notably broccoli, has been implicated in their protective effects. Likewise, the consumption of garlic and its sulphur-containing compounds such as alliin have been associated with a reduction in risk of prostate cancer. In this study, we tested whether consuming glucoraphanin derived from broccoli seeds and alliin derived from garlic resulted in the occurrence of these potential bioactive compounds in the prostate, which may contribute to our understanding of the putative protective effects of these dietary components. We recruited 42 men scheduled for a trans-perineal prostate biopsy into a randomised, double-blinded, 2 × 2-factorial dietary supplement four-week intervention study, and 39 completed the study. The two active interventions were supplements providing glucoraphanin from broccoli (BroccoMax®) and alliin from garlic (Kwai Heartcare®). Following the intervention, prostate biopsy tissue was analysed for the presence of sulforaphane and its thiol conjugates and for alliin and associated metabolites. Sulforaphane occurred in significantly higher levels in the prostate tissue (both within the transition and peripheral zone) of men consuming the glucoraphanin containing supplements (p < 0.0001) compared to men not consuming these supplements. However, while alliin and alliin-derived metabolites were detected within the prostate, there was no significant difference in the concentrations of these compounds in the prostate of men consuming supplements derived from garlic compared to men not consuming these supplements.

Biochemical Characterization of a Novel Myrosinase Rmyr from Rahnella inusitata for High-Level Preparation of Sulforaphene and Sulforaphane.

Myrosinase is a biotechnological tool for the preparation of sulforaphane and sulforaphene with a variety of excellent biological activities. In this study, a gene encoding the novel glycoside hydrolase family 3 (GH3) myrosinase Rmyr from Rahnella inusitata was heterologously expressed in Escherichia coli BL21 (DE3). The purified Rmyr shows the highest activity at 40 °C and pH 7.0; meanwhile, its half-life at 30 °C reaches 12 days, indicating its excellent stability. Its sinigrin-, glucoraphenin-, and glucoraphanin-hydrolyzing activities were 12.73, 4.81, and 6.99 U/mg, respectively. Rmyr could efficiently degrade the radish seed-derived glucoraphenin and the broccoli seed-derived glucoraphanin into sulforaphene and sulforaphane within 10 min with the highest yields of 5.07 mg/g radish seeds and 9.56 mg/g broccoli seeds, respectively. The highest conversion efficiencies of sulforaphane from glucoraphanin and sulforaphene from glucoraphenin reached up to 92.48 and 97.84%, respectively. Therefore, Rmyr is a promising and potent biocatalyst for efficient and large-scale preparation of sulforaphane and sulforaphene.

Approaches for enhancing the stability and formation of sulforaphane.

The isothiocyanate sulforaphane (SF) is one of the most potent naturally occurring Phase 2 enzymes inducers derived from brassica vegetables like broccoli, cabbage, brussel sprouts, etc. Ingestion of broccoli releases SF via hydrolysis of glucoraphanin (GRP) by plant myrosinase and/or intestinal microbiota. However, both SF and plant myrosinase are thermal-labile, and the epithiospecifier protein (ESP) directs the hydrolysis of GRP toward formation of sulforaphane nitrile instead of SF. In addition, bacterial myrosinase has low hydrolyzing efficiency. In this review, we discuss strategies that could be employed to improve the stability of SF, increase SF formation during thermal and non-thermal processing of broccoli, and enhance the myrosinase-like activity of the gut microbiota. Furthermore, new cooking methods or blanching technologies should be developed to maintain myrosinase activity, and novel thermostable myrosinase and/or microbes with high SF producing abilities should also be developed.

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.

Elevated CO2 improves glucosinolate metabolism and stimulates anticancer and anti-inflammatory properties of broccoli sprouts.

Sprouting process enhances plant bioactive compounds. Broccoli (Brassica oleracea L) sprouts are well known for their high levels of glucosinolates (GLs), amino acids, and antioxidants, which offer outstanding biological activities with positive impacts on plant metabolism. Elevated CO2 (eCO2, 620 ppm) was applied for 9 days to further improve nutritive and health-promoting values of three cultivars of broccoli sprouts i.e., Southern star, Prominence and Monotop. eCO2 improved sprouts growth and induced GLs accumulation e.g., glucoraphanin, possibly through amino acids production e.g., high methionine and tryptophan. There were increases in myrosinase activity, which stimulated GLs hydrolysis to yield health-promoting sulforaphane. Interestingly, low levels of ineffective sulforaphane nitrile were detected and positively correlated with reduced epithiospecifier protein after eCO2 treatment. High glucoraphanin and sulforaphane levels in eCO2 treated sprouts improved the anticarcinogenic and anti-inflammatory properties of their extracts. In conclusion, eCO2 treatment enriches broccoli sprouts with health-promoting metabolites and bioactivities.

Effect of melatonin treatment on visual quality and health-promoting properties of broccoli florets under room temperature.

Effect of melatonin treatment on visual quality and contents of health-promoting compounds of broccoli florets under room temperature was investigated in the present study. Broccoli florets were treated with 1 µM melatonin and then stored at room temperature. Results showed that melatonin treatment could delay the post-harvest senescence of broccoli, and performed well in maintaining higher levels of antioxidants, such as carotenoids, vitamin C and total phenols, as well as higher antioxidant capacity than the control. Besides, 1 µM melatonin treatment sustained higher content of glucosinolates, and also resulted in increased percentage of the most potent anticarcinogenic profile, glucoraphanin. Further analysis revealed that 1 µM melatonin strongly induced the expression of glucosinolate biosynthesis-related genes BoMYB28, BoMYB34, BoCYP79F1, and BoCYP79B2, as well as BoTGG1, a gene involved in glucosinolate hydrolysis. In conclusion, post-harvest treatment with 1 µM melatonin is potential in maintaining visual quality and health-promoting properties of broccoli florets.

Differential expression of major genes involved in the biosynthesis of aliphatic glucosinolates in intergeneric Baemoochae (Brassicaceae) and its parents during development.

KEY MESSAGE: Thus study found the temporal and spatial relationship between production of aliphatic glucosinolate compounds and the expression profile of glucosinolate-related genes during growth and development in radish, Chinese cabbage, and their intergeneric hybrid baemoochae plants. Glucosinolates (GSLs) are one of major bioactive compounds in Brassicaceae plants. GSLs play a role in defense against microbes as well as chemo-preventative activity against cancer, which draw attentions from plant scientists. We investigated the temporal relationship between production of aliphatic Glucosinolate (GSLs) compounds and the expression profile of GSL related genes during growth and development in radish, Chinese cabbage, and their intergeneric hybrid, baemoochae. Over the complete life cycle, Glucoraphasatin (GRH) and glucoraphanin (GRE) predominated in radish, whereas gluconapin (GNP), glucobrassicanapin (GBN), and glucoraphanin (GRA) abounded in Chinese cabbage. Baemoochae contained intermediate levels of all GSLs studied, indicating inheritance from both radish and Chinese cabbage. Expression patterns of BCAT4, CYP79F1, CYP83A1, UGT74B1, GRS1, FMOgs-ox1, and AOP2 genes showed a correlation to their corresponding encoded proteins in radish, Chinese cabbage, and baemoochae. Interestingly, there is a sharp change in gene expression pattern involved in side chain modification, particularly GRS1, FMOgs-ox1, and AOP2, among these plants during the vegetative and reproductive stage. For instance, the GRS1 was strongly expressed during leaf development, while both of FMOgs-ox1 and AOP2 was manifested high in floral tissues. Furthermore, expression of GRS1 gene which is responsible for GRH production was predominantly expressed in leaf tissues of radish and baemoochae, whereas it was only slightly detected in Chinese cabbage root tissue, explaining why radish has an abundance of GRH compared to other Brassica plants. Altogether, our comprehensive and comparative data proved that aliphatic GSLs biosynthesis is dynamically and precisely regulated in a tissue- and development-dependent manner in Brassicaceae family members.

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.

Profiling glucosinolate metabolites in human urine and plasma after broccoli consumption using non-targeted and targeted metabolomic analyses.

Broccoli is a popular brassica vegetable and its consumption may decrease the occurrence of cancer in certain populations. To gain insight into the metabolites that may induce physiological responses to broccoli intake, a non-targeted metabolomic approach and a targeted approach for analysis of glucosinolate metabolites were developed using high resolution accurate mass spectrometry. A human study was conducted in which 6 subjects consumed a single meal of 200 g of uncooked broccoli florets. The metabolomic analysis revealed changes in endogenous metabolites and a decrease in hippuric acid after broccoli consumption. Targeted analysis using high-resolution, accurate mass-mass spectrometry (HRAM-MS) enabled detection of low concentrations (nM) of glucosinolate metabolites in human urine and plasma. Glucosinolate metabolites were found in human urine (13) and plasma (8), respectively. Metabolites from methoxyl-indole glucosinolates, arising from broccoli consumption, are reported for the first time. Most glucosinolate metabolites reached their peak concentration in urine 2-4 h after consumption while, in plasma, peak maxima were achieved 2 h after intake. The results suggest that glucoraphanin metabolites (sulforaphane, sulforaphane cysteine, sulforaphane N-acetyl cysteine) and indole metabolites (ascorbigen and methoxyl ascorbigen from indole glucosinolates) may serve as marker compounds for the intake of broccoli.

Accumulation of Dietary S-Methyl Cysteine Sulfoxide in Human Prostate Tissue.

SCOPE: Observational studies have associated consumption of cruciferous vegetables with reduced risk of prostate cancer. This effect has been associated with the degradation products of glucosinolates-thioglycosides that accumulate within crucifers. The possible role of S-methyl cysteine sulfoxide, a metabolite that also accumulates in cruciferous vegetables, and its derivatives, in cancer prevention is relatively unexplored compared to glucosinolate derivatives. The hypothesis that consuming a broccoli soup results in the accumulation of sulfate (a SMCSO derivative) and other broccoli-derived metabolites in prostate tissue is tested. METHODS AND RESULTS: Eighteen men scheduled for transperineal prostate biopsy were recruited into a 4-week parallel single blinded diet supplementation study (NCT02821728). Nine men supplemented their diet with three 300 mL portions of a broccoli soup each week for four weeks prior to surgery. Analyses of prostate biopsy tissues reveal no detectable levels of glucosinolates and derivatives. In contrast, SMCSO is detected in prostate tissues of the participants, with significantly higher levels in tissue of men in the supplementation arm. SMCSO was also found in blood and urine samples from a previous intervention study with the identical broccoli soup. CONCLUSION: The consequences of SMCSO accumulation in prostate tissues and its potential role in prevention of prostate cancer remains to be investigated.

Long-Term Intake of Glucoraphanin-Enriched Kale Suppresses Skin Aging via Activating Nrf2 and the TßRII/Smad Pathway in SAMP1 Mice.

Sulforaphane, a potent antioxidant compound, is unstable at ambient temperature, whereas its precursor glucoraphanin is stable and metabolized to sulforaphane. Thus, we hypothesized that glucoraphanin-rich diet could effectively induce antioxidant enzyme activities and investigated the protective effects of long-term intake of a glucoraphanin-enriched kale (GEK) diet on skin aging in senescence-accelerated mouse prone 1 (SAMP1) mice. The senescence grading score was significantly lower after treatment with GEK for 39 weeks than that of the control mice. GEK also suppressed the thinning of the dorsal skin layer. Moreover, the GEK treatment enhanced the collagen production and increased the nuclear translocation of Nrf2 and HO-1 expression level in the skin tissue. TßRII and Smad3 expressions were clearly higher in the GEK-treated group than in the control group. Thus, GEK suppressed senescence in SAMP1 mice by enhancing the antioxidant activity and collagen production via the TßRII/Smad3 pathway, suggesting its practical applications for protection against skin aging.

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.

Bioavailability of Sulforaphane Following Ingestion of Glucoraphanin-Rich Broccoli Sprout and Seed Extracts with Active Myrosinase: A Pilot Study of the Effects of Proton Pump Inhibitor Administration.

We examined whether gastric acidity would affect the activity of myrosinase, co-delivered with glucoraphanin (GR), to convert GR to sulforaphane (SF). A broccoli seed and sprout extract (BSE) rich in GR and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor. Gastric acidity appears to attenuate GR bioavailability, as evidenced by more SF and its metabolites being excreted after participants started taking omeprazole. Enteric coating enhanced conversion of GR to SF, perhaps by sparing myrosinase from the acidity of the stomach. There were negligible effects of age, sex, ethnicity, BMI, vegetable consumption, and bowel movement frequency and quality. Greater body mass correlated with reduced conversion efficiency. Changes in the expression of 20 genes in peripheral blood mononuclear cells were evaluated as possible pharmacodynamic indicators. When grouped by their primary functions based on a priori knowledge, expression of genes associated with inflammation decreased non-significantly, and those genes associated with cytoprotection, detoxification and antioxidant functions increased significantly with bioavailability. Using principal components analysis, component loadings of the changes in gene expression confirmed these groupings in a sensitivity analysis.

Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea.

KEY MESSAGE: Overexpression of BoMYB29 gene up-regulates the aliphatic glucosinolate pathway in Brassica oleracea plants increasing the production of the anti-cancer metabolite glucoraphanin, and the toxic and pungent sinigrin. Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties.

Transcriptional changes in prostate of men on active surveillance after a 12-mo glucoraphanin-rich broccoli intervention-results from the Effect of Sulforaphane on prostate CAncer PrEvention (ESCAPE) randomized controlled trial.

BACKGROUND: Epidemiological evidence suggests that consumption of cruciferous vegetables is associated with reduced risk of prostate cancer progression, largely attributed to the biological activity of glucosinolate degradation products, such as sulforaphane derived from glucoraphanin. Because there are few therapeutic interventions for men on active surveillance for prostate cancer to reduce the risk of cancer progression, dietary approaches are an appealing option for patients. OBJECTIVE: We evaluated whether consumption of a glucoraphanin-rich broccoli soup for 1 y leads to changes in gene expression in prostate tissue of men with localized prostate cancer. METHODS: Forty-nine men on active surveillance completed a 3-arm parallel randomized double-blinded intervention study for 12 mo and underwent transperineal template biopsy procedures and dietary assessment at the start and end of the study. Patients received a weekly 300 mL portion of soup made from a standard broccoli (control) or from 1 of 2 experimental broccoli genotypes with enhanced concentrations of glucoraphanin, delivering 3 and 7 times that of the control, respectively. Gene expression in tissues from each patient obtained before and after the dietary intervention was quantified by RNA sequencing followed by gene set enrichment analyses. RESULTS: In the control arm, there were several hundred changes in gene expression in nonneoplastic tissue during the 12 mo. These were associated with an increase in expression of potentially oncogenic pathways including inflammation processes and epithelial-mesenchymal transition. Changes in gene expression and associated oncogenic pathways were attenuated in men on the glucoraphanin-rich broccoli soup in a dose-dependent manner. Although the study was not powered to assess clinical progression, an inverse association between consumption of cruciferous vegetables and cancer progression was observed. CONCLUSION: Consuming glucoraphanin-rich broccoli soup affected gene expression in the prostate of men on active surveillance, consistent with a reduction in the risk of cancer progression. This trial was registered at clinicaltrials.gov as NCT01950143.

Fermentation-based biotransformation of glucosinolates, phenolics and sugars in retorted broccoli puree by lactic acid bacteria.

This study investigated the effect of lactic acid bacteria (LAB) fermentation on the chemical profile of autoclaved broccoli puree, using 7 broccoli-derived LAB isolates (named F1-F5, BF1 and BF2). The total concentrations of glucosinolates (glucoiberin, progoitrin and glucoraphanin) and 10 major phenolics significantly increased from trace level and 289 µg total phenolics/g dry weight (DW) respectively in autoclaved broccoli to 55 to ∼359 µg/g DW and 903 to ∼3105 µg/g DW respectively in LAB fermented broccoli puree. Differential impacts of LAB isolates on the chemical composition of autoclaved broccoli were observed, with the major differences being the significant increase in phloretic acid after fermentation by F1-F5 and an elevated glucoraphanin level in ferments by F1 and BF2. LAB fermentation is a promising way to increase the content of glucosinolates and polyphenolic compounds in broccoli, making the ferments attractive for use as functional ingredients or as a whole functional food.

Transcriptome reveals the gene expression patterns of sulforaphane metabolism in broccoli florets.

Sulforaphane is a new and effective anti-cancer component that is abundant in broccoli. In the past few years, the patterns of variability in glucosinolate content and its regulation in A. thaliana have been described in detail. However, the diversity of glucosinolate and sulforaphane contents in different organs during vegetative and reproductive stages has not been clearly explained. In this paper, we firstly investigated the transcriptome profiles of the developing buds and leaves at bolting stage of broccoli (B52) to further assess the gene expression patterns involved in sulforaphane synthesis. The CYP79F1 gene, as well as nine other genes related to glucorahpanin biosynthesis, MAM1, MAM3, St5b-2, FMO GS-OX1, MY, AOP2, AOP3, ESP and ESM1 were selected by digital gene expression analysis and were validated by quantitative real-time PCR (qRT-PCR). Meanwhile, the compositions of glucosinolates and sulforaphane were detected for correlation analysis with related genes. Finally the RNA sequencing libraries generated 147 957 344 clean reads, and 8 539 unigene assemblies were produced. In digital result, only CYP79F1, in the glucoraphanin pathway, was up-regulated in young buds but absent from the other organs, which was consistent with the highest level of sulforaphane content being in this organ compared to mature buds, buds one day before flowering, flowers and leaves. The sequencing results also presented that auxin and cytokinin might affect glucoraphanin accumulation. The study revealed that up-regulated expression of CYP79F1 plays a fundamental and direct role in sulforaphane production in inflorescences. Two genes of MAM1 and St5b-2 could up-regulated glucoraphanin generation. Synergistic expression of MAM1, MAM3, St5b-2, FMO GS-OX1, MY, ESP and ESM1 was found in sulforaphane metabolism. This study will be beneficial for understanding the diversity of sulforaphane in broccoli organs.

Cross-Linking Antibodies to Beads Using Dimethyl Pimelimidate (DMP).

This protocol describes the cross-linking of antibodies to either Protein A or G agarose beads using dimethyl pimelimidate (DMP). DMP contains an imidoester at each end of a 7-carbon spacer arm and forms an amidine bond with amino groups at alkaline pH; however, cross-linking is more efficient when performed at pH >8. DMP will react with primary amines; thus, it is important that the cross-linking procedure is conducted using nonamine-containing buffers. Following the antibody-bead incubation, beads are washed in Borate buffer to remove residual amines from the Tris buffer. After completion of the cross-linking process in the presence of DMP, unreacted DMP is quenched with ethanolamine, and beads are washed extensively to remove residual noncross-linked antibody before immediate use or storage at 4°C.

Slightly Acidic Electrolyzed Water Treatment Enhances the Main Bioactive Phytochemicals Content in Broccoli Sprouts via Changing Metabolism.

Changes in the content of bioactive phytochemicals in the broccoli sprouts subjected to different slightly acidic electrolyzed water (SAEW) treatments were investigated in the present study. The highest sulforaphane amount in broccoli sprouts treated with SAEW with an available chlorine concentration (ACC) of 50 mg/L was 11.49 mg/g in dry weight (DW), which increased by 61.2% compared to the control. SAEW treatment enhanced the sulforaphane content mainly by increasing the glucoraphanin (GRA) concentration due to the promotion of methionine metabolism and increased myrosinase activities. In addition, the relative anthocyanin contents of light-germinated broccoli under SAEW 50 treatment were 2.03 times that of broccoli sprouts with tap water treatment, and these contents were associated with an increase in phenylalanine ammonia lyase (PAL) activities and phenylalanine participation in biosynthesis. In summary, SAEW promotes metabolism to induce the accumulation of bioactive compounds in broccoli sprouts.