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.

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.

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.

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.

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.

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.

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.

Purification and characterization of aspartic protease derived from Sf9 insect cells.

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS-PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K(m) of 0.85 µM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) µM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM.

Prorenin processing enzyme (PPE) produced by Baculovirus-infected Sf-9 insect cells: PPE is the cysteine protease encoded in the acMNPV gene.

In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610-2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.

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.

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 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.

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.

Characterization and kinetic analysis of enzyme-substrate recognition by three recombinant lactococcal tripeptidases.

Tripeptidases from Lactococcus lactis subsp. lactis (L9PepTR), L. lactis subsp. cremoris (L6PepTR), and L. lactis subsp. hordniae (hTPepTR) were cloned, overexpressed, purified, and characterized. Although these enzymes contained three to seven naturally occurring amino acid differences, both metal-binding and catalytic sites were highly conserved. The k(cat) values of hTPepTR were approximately 1.5- to 2-fold higher than those of L9PepTR, while, for L6PepTR, they were approximately 0.8- to 1.4-times the L9PepTR values. The K(m) of tripeptidase from subsp. lactis (L9PepTR) was considerably larger when glycine was the amino acid located at both the N- and C-terminus of the peptide substrate. In addition, the K(m) values of L9PepTR increased in the following order for YGG, LGG, FGG, SGG, and alpha-aminoisobutyrylglycylglycine, while the k(cat)/K(m) decreased in the same order. These results suggest that the dipole moment and steric hindrance of the N-terminal amino acid side chain may be the most important factors controlling substrate specificity.