Formation, immunomodulatory activities, and enhancement of glucosinolates and sulforaphane in broccoli sprouts: a review for maximizing the health benefits to human.

Broccoli sprouts have been considered as functional foods which have received increasing attention because they have been highly prized for glucosinolates, phenolics, and vitamins in particular glucosinolates. One of hydrolysates-sulforaphane from glucoraphanin is positively associated with the attenuation of inflammatory, which could reduce diabetes, cardiovascular and cancer risk. In recent decades, the great interest in natural bioactive components especially for sulforaphane promotes numerous researchers to investigate the methods to enhance glucoraphanin levels in broccoli sprouts and evaluate the immunomodulatory activities of sulforaphane. Therefore, glucosinolates profiles are different in broccoli sprouts varied with genotypes and inducers. Physicochemical, biological elicitors, and storage conditions were widely studied to promote the accumulation of glucosinolates and sulforaphane in broccoli sprouts. These inducers would stimulate the biosynthesis pathway gene expression and enzyme activities of glucosinolates and sulforaphane to increase the concentration in broccoli sprouts. The immunomodulatory activity of sulforaphane was summarized to be a new therapy for diseases with immune dysregulation. The perspective of this review served as a potential reference for customers and industries by application of broccoli sprouts as a functional food and clinical medicine.

ACE2-like enzyme B38-CAP suppresses abdominal sepsis and severe acute lung injury.

Angiotensin-converting enzyme 2 (ACE2) is the carboxypeptidase to degrade angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and improves the pathologies of cardiovascular disease and acute respiratory distress syndrome (ARDS)/acute lung injury. B38-CAP is a bacteria-derived ACE2-like carboxypeptidase as potent as human ACE2 and ameliorates hypertension, heart failure and SARS-CoV-2-induced lung injury in mice. Recombinant B38-CAP is prepared with E. coli protein expression system more efficiently than recombinant soluble human ACE2. Here we show therapeutic effects of B38-CAP on abdominal sepsis- or acid aspiration-induced acute lung injury. ACE2 expression was downregulated in the lungs of mice with cecal ligation puncture (CLP)-induced sepsis or acid-induced lung injury thereby leading to upregulation of Ang II levels. Intraperitoneal injection of B38-CAP significantly decreased Ang II levels while upregulated angiotensin 1-7 levels. B38-CAP improved survival rate of the mice under sepsis. B38-CAP suppressed the pathologies of lung inflammation, improved lung dysfunction and downregulated elevated cytokine mRNA levels in the mice with acute lung injury. Thus, systemic treatment with an ACE2-like enzyme might be a potential therapeutic strategy for the patients with severe sepsis or ARDS.

ACE2-like carboxypeptidase B38-CAP protects from SARS-CoV-2-induced lung injury.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients.

Rheological, structural and physicochemical characteristics of heat-induced egg albumin/sesbania gum mixed gels.

The effects of sesbania gum (SG) on the rheological and physicochemical properties of heat-induced egg albumin gels were investigated. The formation of the gels of egg albumin (4%, w/v)/SG (0, 0.1, 0.2 and 0.3%, w/v) mixtures were induced by heating. It was evident from the appearance of gels' microstructure that phase separation occurred in egg albumin/SG systems. Non-Newtonian pseudoplastic flow behavior was observed in all the gels and it was further evident that 0.1% of SG could significantly (p < 0.05) improve the apparent viscosity of the egg albumin gels. The rheological characteristics of the gels demonstrated a more stable and plastic character gel formed at 0.1% SG. The temperature stability test showed that the structures of egg albumin/SG gels maintain their stability when reheated. The hardness of the egg albumin gels was improved with the addition of 0.1% SG, and their water holding capacity (WHC) was also improved. For egg albumin gel with 0.1% SG, a three-dimensional stranded network was observed, indicating the formation of a more stable and tight gel. The FTIR results confirmed that egg albumin and SG are thermodynamically incompatible, which suggested that there is no covalent bonding between egg albumin and SG.

Isolation, physical, structural characterization and in vitro prebiotic activity of a galactomannan extracted from endosperm splits of Chinese Sesbania cannabina seeds.

The purpose of this study was to identify and determine the physical and structural characterization of the water-soluble galactomannan extracted from endosperm splits of Chinese S. cannabina seeds. The Sesbania galactomannan (SP) was extracted and purified using a novel method with a high yield (40.3 ± 7.2%). The molecular structure of SP was determined by monosaccharide composition, FTIR and NMR spectroscopy. The structural data showed that SP was galactomannan which composed by a ß-(1/4)-linked mannose backbone with galactopyranosyl residues attached through α-(1/6) linkages. The constant mannose/galactose (M/G) ratio and average molecular weight (Mw) of SP was 1.6:1 and 2.16 × 105 g/mol, respectively. The physical results revealed that SP had many branches on the backbone and existed as a random coil state in aqueous solution. SP was a good biopolymer which had smooth and clearer surface with homogeneous composition, and had some degree of crystallinity and prebiotic activity. As a consequence, SP could be a potential prebiotic and was expected to be suitable for applications in food, pharmaceutical, biomedical or cosmetic industries as a promising new biomaterial.

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

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.

Effects of sodium carbonate and potassium carbonate on colloidal properties and molecular characteristics of konjac glucomannan hydrogels.

When konjac glucomannan (KGM) molecules are deacetylated under alkaline conditions, the aqueous KGM solution is transformed into a thermally stable gel. In this study, series of Na2CO3-induced and K2CO3-induced KGM hydrogels were prepared by deacetylation using different concentrations (0.1, 0.2, 0.3, and 0.4 M) of alkali. The hydrogels were characterized using texture profile analysis, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, X-ray diffraction, and rheological property analysis. The data showed that KGM hydrogel formation was facilitated at all the alkali concentrations used. The mechanisms of Na2CO3-induced and K2CO3-induced KGM hydrogels formation differed slightly. The hardness, springiness, chewiness, gumminess, and storage modulus G' of the Na2CO3-induced KGM hydrogels initially increased and then decreased with increasing alkali concentration. However, the values of the corresponding properties of the K2CO3-induced KGM hydrogels increased with increasing alkali concentration. All the data were consistent with the structures observed using SEM. The 0.3 M Na2CO3-induced KGM hydrogel had the highest hardness and storage modulus G', a well-proportioned network structure, and a dense architecture; 0.3 M Na2CO3 was therefore the most suitable modifier for inducing KGM hydrogel formation.

Cloning and characterization of the novel d-aspartyl endopeptidase, paenidase, from Paenibacillus sp. B38.

Paenidase is the first micro-organism-derived d-aspartyl endopeptidase that specifically recognizes an internal d-Asp residue to cleave [d-Asp]-X peptide bonds. Using peptide sequences obtained from the protein, we performed PCR with degenerate primers to amplify the paenidase I-encoding gene. Nucleotide sequencing revealed that mature paenidase I consist of 322 amino acid residues and that the protein is encoded as a pro-protein with a 197-amino-acid N-terminal extension compared to the mature protein. Paenidase I exhibits amino acid sequence similarity to several penicillin-binding proteins. In addition, paenidase I was classified into peptidase family S12 based on a MEROPS database search. Family S12 contains serine-type d-Ala-d-Ala carboxypeptidases that have three active site residues (Ser, Lys and Tyr) in the conserved motifs Ser-Xaa-Thr-Lys and Tyr-Xaa-Asn. These motifs were conserved in the primary structure of paenidase I, and the role of these residues was confirmed by site-directed mutagenesis.

Effect of slightly acidic electrolyzed water on bioactive compounds and morphology of broccoli sprouts.

The producers of broccoli sprouts have become increasingly interested in improving their sulforaphane content. This study has evaluated the effects of slightly acidic electrolyzed water (SAEW) with different available chlorine concentrations (ACC) on broccoli sprouts: their content of some bioactive compounds such as glucosinolates, their morphology, and their total bacterial counts. The results have shown that SAEW might affect the content of sulforaphane by influencing the content of glucosinolates and the activity of myrosinase. SAEW inhibited the growth of broccoli sprouts: their fresh weight decreased as the available chlorine concentration (ACC) of the SAEW increased, but the different solutions did not affect their dry weight. The number of microorganisms on the broccoli sprout decreased by 1.71logCFU/g after using the SAEW with ACC value of 50mg/L treatment compared with tap water treatment. Overall, although SAEW adversely affected the morphology of broccoli sprouts, with a suitable ACC it can be a useful tool for enhancing the amount of secondary metabolites and reducing the microbial counts on broccoli sprouts intended for fresh consumption as a functional food.

Physicochemical changes in myofibrillar proteins extracted from pork tenderloin thawed by a high-voltage electrostatic field.

The application of a high-voltage electrostatic field (HVEF) is a novel method for thawing. To determine the effects of HVEF thawing (voltage range: -25kV to 0kV) on myofibrillar protein oxidation and/or denaturation and to provide a theoretical understanding of this process, pork tenderloin was thawed by traditional and HVEF methods. Based on the total sulfhydryl and carbonyl contents, further protein oxidation did not occur during HVEF thawing. It was proposed that the free radical-mediated oxidation of myofibrillar proteins was hindered by HVEF. The results of dynamic rheological analysis, protein aggregation and gel texture studies showed that HVEF thawing, especially -10kV HVEF thawing, led to better indicators than those achieved under air thawing. A higher abundance of proteins extracted from -10kV HVEF-thawed samples compared with air-thawed samples was found. Finally, this study showed that thawing under -10kV conditions did not affect the structure of myofibrillar proteins.

Effects of dough mixing time before adding konjac glucomannan on the quality of noodles.

In this study, effects of 5% konjac glucomannan (KGM) blended with low-protein flour at different dough mixing duration on the properties of dough and noodles were investigated. To prepare the KGM noodle samples, 5% KGM was added after low-protein flour mixed with water for 0, 2 and 4 min, respectively. The three samples above were defined as T0 KGM, T2 KGM and T4 KGM noodle samples, respectively. The results revealed that the elastic modulus (G') and viscous modulus (G″) of dough both increased with extending dough mixing time before adding KGM. T4 KGM samples showed the least cooking loss. Textural properties including hardness, cohesiveness and tensile strength of KGM noodles had a tendency to increase with a longer dough mixing time before adding KGM. Microstructure of dough and noodles confirmed that a longer dough mixing time before adding KGM made microstructure more compact with a thickened gluten matrix. The sensory quality of the T2 KGM and T4 KGM samples was better than that of the T0 KGM samples.

Effects of konjac glucomannan on heat-induced changes of wheat gluten structure.

Effects of konjac glucomannan on the structure of wheat gluten were investigated at variable temperatures in this study. Dynamic oscillatory rheology study showed that konjac glucomannan conferred stiffness on gluten with a higher tan δ data at 25°C and 55°C, but this parameter was lower at 75°C and 95°C. Konjac glucomannan decreased the content of thiol equivalent groups and increased the α-helix/ß-sheet content ratio, respectively. The thicker layer of gluten protein with 5% konjac glucomannan was observed by scanning electron microscopy. This study revealed that konjac glucomannan could alter the conformations of gluten proteins upon heating via non-covalent interactions and physical entanglements. It is likely that konjac glucomannan promotes protein aggregation by strengthening hydrophobic interaction at 25°C and 55°C, and alleviates heat-induced denaturation by decreasing the flexibility of polypeptide chain at higher 75°C and 95°C.

Interactions of ß-Conglycinin (7S) with Different Phenolic Acids-Impact on Structural Characteristics and Proteolytic Degradation of Proteins.

p-Coumalic acid (PCA), caffeic acid (CA), gallic acid (GA) and chlorogenic acid (CGA) are the major phenolic acids that co-exist with soy protein components in foodstuffs. Surprisingly, there are only a handful of reports that describe their interaction with ß-Conglycinin (7S), a major soy protein. In this report, we investigated the interaction between phenolic acids and soy protein 7S and observed an interaction between each of these phenolic acids and soy protein 7S, which was carried out by binding. Further analysis revealed that the binding activity of the phenolic acids was structure dependent. Here, the binding affinity of CA and GA towards 7S was found to be stronger than that of PCA, because CA and GA have one more hydroxyl group. Interestingly, the binding of phenolic acids with soy protein 7S did not affect protein digestion by pepsin and trypsin. These findings aid our understanding of the relationship between different phenolic acids and proteins in complex food systems.

The protective role of protein L-isoaspartyl (D-aspartate) O-methyltransferase for maintenance of mitochondrial morphology in A549 cell.

PURPOSE: The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells. MATERIALS AND METHODS: We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1. RESULTS: We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content. CONCLUSIONS: The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.

Intracellular acidification is required for full activation of the sweet taste receptor by miraculin.

Acidification of the glycoprotein, miraculin (MCL), induces sweet taste in humans, but not in mice. The sweet taste induced by MCL is more intense when acidification occurs with weak acids as opposed to strong acids. MCL interacts with the human sweet receptor subunit hTAS1R2, but the mechanisms by which the acidification of MCL activates the sweet taste receptor remain largely unexplored. The work reported here speaks directly to this activation by utilizing a sweet receptor TAS1R2 + TAS1R3 assay. In accordance with previous data, MCL-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of MCL was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of MCL was proportional to intracellular pH regardless of types of acids. These results suggest that intracellular acidity is required for full activation of the sweet taste receptor by MCL.

New insights into the antioxidant activity and components in crude oat oil and soybean oil.

Developing new antioxidants and using natural examples is of current interest. This study evaluated the antioxidant activities and the ability to inhibit soybean oil oxidation of oat oil obtained with different solvents. Oat oil extract obtained by ethanol extraction gave the highest antioxidant activity with a DPPH radical (1,1-diphenyl-2-picrylhydrazyl) scavenging activity of 88.2 % and a reducing power (A 700) of 0.83. Oat oil extracted by ethanol contained the highest polyphenol and α-tocopherol content. Significant correlation was observed between the total polyphenol contents, individual phenolic acid, α-tocopherol, and DPPH radical scavenging activity. Soybean oil with 2 % added oat oil showed low malondialdehyde content (8.35 mmol mL(-1)), suggesting that the added oat oil inhibited oxidation. Oat oil showed good antioxidant activity, especially when extracted with ethanol which could also retard the oxidation of soybean oil . DPPH radical scavenging activity was the best method to evaluate the antioxidant activity and components of oat oil.

Inhibition of the DNA polymerase and RNase H activities of HIV-1 reverse transcriptase and HIV-1 replication by Brasenia schreberi (Junsai) and Petasites japonicus (Fuki) components.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) possesses two distinct enzymatic activities: those of RNA- and DNA-dependent DNA polymerases and RNase H. In the current HIV-1 therapy, all HIV-1 RT inhibitors inhibit the activity of DNA polymerase, but not that of RNase H. We previously reported that ethanol and water extracts of Brasenia schreberi (Junsai) inhibited the DNA polymerase activity of HIV-1 RT [Hisayoshi et al. (2014) J Biol Macromol 14:59-65]. In this study, we screened 43 edible plants and found that ethanol and water extracts of Brasenia schreberi and water extract of Petasites japonicus strongly inhibit not only the activity of DNA polymerase to incorporate dTTP into poly(rA)-p(dT)15 but also the activity of RNase H to hydrolyze the RNA strand of an RNA/DNA hybrid. In addition, these three extracts inhibit HIV-1 replication in human cells, with EC50 values of 1-2 µg/ml. These results suggest that Brasenia schreberi and Petasites japonicus contain substances that block HIV-1 replication by inhibiting the DNA polymerase activity and/or RNase H activity of HIV-1 RT.

Effect of a small amount of sodium carbonate on konjac glucomannan-induced changes in wheat starch gel.

Wheat starch gels were produced with konjac glucomannan (KGM) and low concentrations of Na2CO3 (0.1-0.2 wt% of starch) using a rapid viscosity analyzer (RVA). The gelling properties of wheat starch in varying ratios of KGM and Na2CO3 were characterized by differential scanning calorimetry (DSC), rheometry and confocal laser scanning microscopy (CLSM). A small amount of Na2CO3 resulted in gels with increased elasticity whereas structural ordering during retrogradation was insignificantly affected. Comparison of CLSM images of composite gels revealed that Na2CO3 at 0.2 wt% of starch allowed the formation of fiber-like extensions around scattered swollen granules by KGM and amylose interaction, making swollen granules disperse within the micro phase, which was not typical in CLSM images of gels in the absence of Na2CO3. Dynamic storage modulus and dynamic power law exponent were substantially higher than those observed for the same concentration of KGM in the presence of Na2CO3, supporting the hypothesis that Na2CO3 could promote strong interchain associations between KGM and starch components.

Rapid survey of four Asp isomers in disease-related proteins by LC-MS combined with commercial enzymes.

Until relatively recently, it was considered that D-amino acids were excluded from living systems except for the cell wall of microorganisms. However, D-aspartate residues have now been detected in long-lived proteins from various tissues of elderly humans. Formation of D-aspartate in proteins induces aggregation and loss of function, leading to age-related disorders such as cataracts and Alzheimer disease. A recent study used LC-MS to analyze isomers of Asp residues in proteins precisely without complex purification of the proteins. However, to identify the four Asp isomers (Lα, Lß, Dß, and Dα) on the chromatogram, it was necessary to synthesize reference peptides containing the four different Asp isomers as standards. Here, we describe a method for rapidly and comprehensively identifying Asp isomers in proteins using a combination of LC-MS and commercial enzymes without synthesizing reference peptides. The protein sample is treated with trypsin, trypsin plus Asp-N, trypsin plus PIMT, trypsin plus paenidase, and the resulting peptides are applied to LC-MS. Because Asp-N hydrolyzes peptide bonds on the N-terminus of only Lα-Asp residues, it differentiates peptides containing Lα-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing Lß-Asp residues, and paenidase internally cleaves the C-terminus of Dα-Asp residues. This approach was successfully applied to the analysis of all tryptic peptides in aged lens. The comprehensive quantitative data of Asp isomer formation in age-related proteins obtained via this method might be used as biomarkers of age-related disease.