Bidirectional fermentation of Monascus and Mulberry leaves enhances GABA and pigment contents: establishment of strategy, studies of bioactivity and mechanistic.

Bidirectional fermentation is a technology that utilizes fungi to ferment medicinal edible substrates, with synergistic and complementary advantages. In this work, a fermentation strategy was established to produce a high yield of γ-aminobutyric acid (GABA) and Monascus pigments (MPs) using Monascus and mulberry leaves (MLs). Firstly, the basic fermentation parameters were determined using single-factor experiments, followed by Plackett-Burman (PB) experimental design to identify MLs, glucose, peptone, and temperature as significant influencing factors. The fermentation parameters were optimized using an artificial neural network (ANN). Finally, the effects of bidirectional fermentation of MLs and Monascus were investigated by bioactivity analysis, microstructure observation, and RT-qPCR. The outcomes showed that the bidirectional fermentation significantly increased the bioactive content and promoted the secondary metabolism of Monascus. The established fermentation conditions were 44.2 g/L of MLs, 57 g/L of glucose, 15 g/L of peptone, 1 g/L of MgSO4, 2 g/L of KH2PO4, 8% (v/v) of inoculum, 180 rpm, initial pH 6, 32 °C and 8 days. The content of GABA reached 13.95 g/L and the color value of MPs reached 408.07 U/mL. This study demonstrated the feasibility of bidirectional fermentation of MLs and Monascus, providing a new idea for the application of MLs and Monascus.

Combined volatile compounds and non-targeted metabolomics analysis reveals variation in flavour characteristics, metabolic profiles and bioactivity of mulberry leaves after Monascus purpureus fermentation.

BACKGROUND: Mulberry leaves (MLs) are widely used in food because of their nutritional and functional characteristics. However, plant cell walls and natural bitterness influence nutrient release and the flavor properties of MLs. Liquid-state fermentation using Monascus purpureus (LFMP) is a common processing method used to improve food properties. The present study used headspace solid-phase micro extraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) and non-targeted metabolomics to examine changes in volatile and non-volatile metabolites in MLs. The transformation mechanism of LFMP was investigated by microscopic observation and dynamic analysis of enzyme activity, and changes in the biological activity of MLs were analyzed. RESULTS: LFMP significantly increased total phenolics, total flavonoids, free amino acids and soluble sugars in MLs, at the same time as decreasing phytic acid levels. In total, 92 volatile organic compounds (VOCs) were identified and quantified. VOCs such as (2R,3R)-(-)-2,3-butanediol, terpineol and eugenol showed some improvement in the flavour characteristics of MLs. By using non-targeted metabolomics, 124 unique metabolites in total were examined. LFMP altered the metabolic profile of MLs, mainly in plant secondary metabolism, lipid metabolism and amino acid metabolism. Microscopic observation and dynamic analysis of enzyme activity indicated that LFMP promoted cell wall degradation and biotransformation of MLs. In addition, LFMP significantly increased the angiotensin I-converting enzyme and α-glucosidase inhibitory activity of MLs. CONCLUSION: LFMP altered the flavour characteristics, metabolite profile and biological activity of MLs. These findings will provide ideas for the processing of MLs into functional foods. In addition, they also provide useful information for biochemical studies of fermented MLs. © 2023 Society of Chemical Industry.

MicroRNA-125b mediates Interferon-γ-induced downregulation of the vitamin D receptor in systemic lupus erythematosus.

BACKGROUND: The inflammatory factor interferon (IFN)-γ is related to the occurrence and development of systemic lupus erythematosus (SLE). The vitamin D receptor (VDR) has an anti-inflammatory effect and its downregulation is involved in the onset of SLE. Our previous studies have confirmed that the expression of VDR in SLE peripheral blood mononuclear cells (PBMCs) is downregulated, which is negatively correlated with disease activity and inflammation. However, the mechanism underlying VDR downregulation in SLE is unknown. METHODS: Based on the results of computer simulation analysis, the expression of VDR and four microRNAs (miR-17-3p, miR-34a, miR-346, and miR-125b) in SLE PBMC cells was analyzed under proinflammatory cytokine IFN­Î³ treatment, and miR-125b was identified as the target miRNA. The relationship between IFN­Î³, miR-125b, and VDR was further assessed in THP­1 cells. RESULTS: We showed that IFN­Î³ inhibited the expression of VDR and miR-125b. Further study revealed that VDR mRNA was positively correlated with miR-125b in THP­1 cells after IFN­Î³ intervention. After transfection of miR-125b mimic or inhibitor, the expression of VDR in the miR-125b inhibitor group was lower than in the control group and miR-125b mimic group, while expression in the control group was lower than in miR-125b mimic group. Transfection of miR-125b inhibitor into THP­1 cells could further promote the ability of IFN­Î³ to inhibit VDR. CONCLUSION: The decrease in VDR expression promotes development of inflammation and SLE. These data suggest that miR-125b may mediate inflammatory factor IFN-γ-induced downregulation of VDR in the pathogenesis of SLE.

Human umbilical cord-derived mesenchymal stem cells alleviate valproate-induced immune stress and social deficiency in rats.

Introduction: Autism spectrum disorders (ASD) are a set of heterogeneous neurodevelopmental disorders characterized by impaired social interactions and stereotypic behaviors. Current clinical care is palliative at the most and there remains huge unmet medical need to fully address the core symptoms of ASD. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are emerging as a promising candidate for ASD treatment, but the precise mechanism remains controversial. Methods: In vitro studies we performed the transwell migration assay to explore the interaction between hUC-MSCs and the primary-cultured cortical neurons. Then we determined the therapeutic effects of intravenous administration of hUC-MSCs in rats challenged with valproic acid (VPA) during gestation, a well-defined rat model of autism. Results: Our studies showed that hUC-MSCs promoted the growth of primary-cultured cortical neurons. Furthermore, our results demonstrated that hUC-MSCs significantly alleviated microglial activation in the brain, especially in the anterior cingulate cortex, and effectively improved the sociability of the VPA-exposed rats. Discussion: These results offer valuable insights for clinical translation and further research on the mechanisms of hUC-MSCs in psychiatric disorders characterized by microglial activation, particularly in cases of autism, shall be warranted.

Multi-frequency ultrasound-assisted cellulase extraction of protein from mulberry leaf: Kinetic, thermodynamic, and structural properties.

The effects of different extraction methods (traditional extraction, ultrasound extraction, cellulase extraction, and ultrasound-assisted cellulase extraction) on the yield of mulberry leaf protein (MLP) were investigated, and the results revealed that multi-frequency ultrasound-assisted cellulase extraction was the most efficient extraction method. The mechanism of the synergistic extraction method used to efficiently extract protein from mulberry leave was investigated, focusing on the kinetics and thermodynamics of the enzymatic process. The results revealed that kinetic parameters KM decreased by 14.07% and kA increased by 5.02%, and the thermodynamic parameters Ea, ΔH, and ΔS decreased by 44.81%, 48.41%, and 21.12 %, respectively, following the process of multi-frequency ultrasound (MFU) pretreatment. The spectral analysis with fluorescence spectra manifested that ultrasound exposed hydrophobic groups and induced molecular unfolding of MLP. Atomic force microscope showed that ultrasound decreased particle size while increasing flexibility of MLP. The effect of ultrasound increases the binding frequency of cellulase and substrates, resulting in greater affinity between the two and promoting the solubilization of MLP. This study provides a theoretical basis to improve the application prospects of MLP.

Characteristics of enzymolysis of silkworm pupa protein after tri-frequency ultrasonic pretreatment: kinetics, thermodynamics, structure and antioxidant changes.

As a by-product of the sericulture industry, the utilization rate of silkworm pupa resources is currently not high. Proteins are converted into bioactive peptides through enzymatic hydrolysis. Not only can it solve the utilization problem, but it also creates more valuable nutritional additives. Silkworm pupa protein (SPP) was pretreated with tri-frequency ultrasonic (22/28/40 kHz). Effects of ultrasonic pretreatment on enzymolysis kinetics, enzymolysis thermodynamics, hydrolysate structure as well as hydrolysate antioxidant of SPP were investigated. Ultrasonic pretreatment significantly increased the hydrolysis efficiency, showing a 6.369% decrease in k m and a 16.746% increase in k A after ultrasonic action (p < 0.05). The SPP enzymolysis reaction followed a second-order rate kinetics model. Evaluation of enzymolysis thermodynamics revealed that Ultrasonic pretreatment markedly enhanced the SPP enzymolysis, leading to a 21.943% decrease in E a. Besides, Ultrasonic pretreatment significantly increased SPP hydrolysate's surface hydrophobicity, thermal stability, crystallinity, and antioxidant activities (DPPH radical scavenging activity, Fe2+ chelation ability, and reducing power). This study indicated that tri-frequency ultrasonic pretreatment could be an efficient approach to enhancing the enzymolysis and improving the functional properties of SPP. Therefore, tri-frequency ultrasound technology can be applied industrially to enhance enzyme reaction process.

The effects of CPBL + SBAR teaching mode among the nursing students.

OBJECTIVE: To investigate the effect of CPBL (Problem Based Learning on Case) combined with SBAR (Situation, Background, Assessment and Recommendation) mode in clinical teaching for nursing students. BACKGROUND: Clinical internship training is the key step for the nursing education. The clinical thinking ability of Chinese nurses is mostly at the middle or low level. To improve clinical thinking ability, especially critical thinking, is of great significance to the nursing practice education. METHODS: 102 nursing students of the Third Xiangya Hospital, Central South University were selected as the participants from June 2018 to September 2019. We applied the CPBL + SBAR mode to the participants during the practice teaching, and conducted questionnaires and semi-structured interview pre- and post-intervention. RESULTS: After intervention, the nursing students' scores of critical thinking ability (311.47 ± 22.67) were higher than those before intervention (289.75 ± 19.94); the total score (69.84 ± 6.217) and scores in four dimensions of positive problem orientation (RPO), negative problem orientation (NPO), avoidance style (AS) and impulse neglect style (ICS) were all lower than that before intervention (75.53 ± 6.09); the score of rational problem solving (RPS) (18.38 ± 2.714) was higher comparing to the that of pre-intervention (17.11 ± 2.20); the difference was statistically significant (P < 0.05). The approval rate of nursing students to the positive effect of CPBL + SBAR teaching mode was from 78.43% to 93.13%. The semi-structured interview suggested that the model was worth promoting, but the implementation duration was limited. CONCLUSION: CPBL + SBAR teaching mode could improve the nursing students' critical thinking and problem-solving ability. The nursing students and teachers' satisfaction to the new teaching mode was high.

Production of ACE inhibitory peptides from sweet sorghum grain protein using alcalase: Hydrolysis kinetic, purification and molecular docking study.

In this study, sweet sorghum grain protein (SSGP) was hydrolyzed using alcalase yielding ACE inhibitory peptides. A kinetic model was proposed to describe the enzymolysis process of SSGP. The kinetic parameters, a and b, were determined according to experimental data. It was found that the model was reliable to describe the kinetic behaviour for SSGP hydrolysis by alcalase. After hydrolysis, the SSGP hydrolysate with DH of 19% exhibited the strongest ACE inhibitory activity and the hydrolysate was then used to isolate ACE inhibitory peptides. A novel ACE inhibitory peptide was successfully purified from this hydrolysate by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reversed-phased high performance liquid chromatography (RP-HPLC). The amino acid sequence of the purified peptide was identified as Thr-Leu-Ser (IC50=102.1 µM). The molecular docking studies revealed that the ACE inhibition of the tripeptide was mainly attributed to its C-terminal Ser, which can effectively interact with the S1 and S2 pockets of ACE. Our studies suggest that the tripeptide from the SSGP hydrolysate can be utilized to develop functional food ingredients or pharmaceuticals for prevention of hypertension.

A novel angiotensin-І converting enzyme (ACE) inhibitory peptide from gastrointestinal protease hydrolysate of silkworm pupa (Bombyx mori) protein: Biochemical characterization and molecular docking study.

Silkworm pupa (Bombyx mori) protein was hydrolyzed using gastrointestinal endopeptidases (pepsin, trypsin and α-chymotrypsin). Then, the hydrolysate was purified sequentially by ultrafiltration, gel filtration chromatography and RP-HPLC. A novel ACE inhibitory peptide, Ala-Ser-Leu, with the IC50 value of 102.15µM, was identified by IT-MS/MS. This is the first report of Ala-Ser-Leu from natural protein. Lineweaver-Burk plots suggest that the peptide is a competitive inhibitor against ACE. The molecular docking studies revealed that the ACE inhibition of Ala-Ser-Leu is mainly attributed to forming very strong hydrogen bonds with the S1 pocket (Ala354) and the S2 pocket (Gln281 and His353). The results indicate that silkworm pupa (B. mori) protein or its gastrointestinal protease hydrolysate could be used as a functional ingredient in auxiliary therapeutic foods against hypertension.

Characterization, antioxidant and antitumor activities of polysaccharides from purple sweet potato.

Three polysaccharides, PSPP1-1, PSPP2-1 and PSPP3-1, were isolated from purple sweet potato. The three polysaccharides belonged to ß-type polysaccharides and contained low proportions of proteins and uronic acids. PSPP1-1 and PSPP3-1 with molecular weights of 33.3 and 75.3 kDa, respectively, were composed of rhamnose, xylose, glucose and galactose, whereas PSPP2-1 with molecular weight of 17.8 kDa was composed of rhamnose and galactose. The three polysaccharides possessed in vitro antioxidant (scavenging DPPH radicals, chelating ferrous ions and reducing power) and antitumor activities (against SGC7901 and SW620 cells) in a dose-dependent manner. Among the three polysaccharides, PSPP2-1 exhibited the strongest reducing power, scavenging activity on DPPH radicals and chelating capability on ferrous ions. PSPP1-1 showed the strongest inhibitory activities on the growth of SGC7901 and SW620 cells. In addition, flow cytometry results showed that PSPP1-1 could induce apoptosis in SGC7901 and SW620 cells. These results suggest that polysaccharides from purple sweet potato are potential natural antioxidant and antitumor agents that can be used as drugs or functional food ingredients.

[On preparation the monoclonal antibody against synthetic antigen of cyclosporine A].

OBJECTIVE: To study the methods for preparing the monoclonal antibody against synthetic antigen of cyclosporine A. METHODS: These included using photochemical reaction to couple cyclosporine A with poly-L-lysine as synthetic antigen, immunizing BALB/c mice, fusing spleen cells and SP2/0 cells. Then hybridoma cells were repeatedly screened and subcloned. The monoclonal antibodies were identified by indirect ELISA. RESULTS: Four hybridoma cells in secreting anti-cyclosporine A monoclonal antibodies were obtained. Only two showed high titer and some specificity. CONCLUSIONS: The methods of antigen synthesis and immunization were practicable. But the methods of antibody preparation and preservation need to be improved.

Rapid detection of Rosa laevigata polysaccharide content by near-infrared spectroscopy.

A method of rapid detecting Rosa laevigata polysaccharide content on the basis of near infrared spectroscopy (NIRS) was established to achieve the purpose of controlling quality of R. laevigata. A total of 129 batches of R. laevigata samples were randomly divided into calibration set and prediction set, number of which were 65 and 64 respectively. The polysaccharide content was measured according to the method provided by Chinese pharmacopoeia, and was 26.05±5.44(%). The Near-infrared diffuse reflectance spectra of R. laevigata were preprocessed by first-order derivative and autoscaling, and was built model with PLS. When 6 Latent variables (LVs) were used in model, the smallest root mean square error of cross validation (RMSECV) was 1.18%, and root mean square error of prediction (RMSEP) was 1.21%. The uninformative variables in spectrum were eliminated by UVE-PLS, and 383 variables were obtained. The prediction accuracy was improved, and RMSECV and RMSEP were 0.90% and 0.99% respectively. Then, 383 variables were further optimized by genetic algorithm (UVE-GA-PLS), and 179 variables were obtained; under this condition, the RMSECV and RMSEP were 0.93% and 1.07% respectively. In this work, result of UVE-PLS was the best. Analyzing variables VIP score in PLS, variable selected by UVE-PLS and UVE-GA-PLS, spectrum region from 7200 cm(-1) to 6700 cm(-1) of the combinations of first and second overtone of C-H and from 5300 cm(-1) to 5000 cm(-1) related to the first overtone of O-H could play an important role in the detection R. laevigata polysaccharide by NIR. Therefore, it was feasible of rapid detection of R. laevigata polysaccharide content by NIRS.

[Determination of angiotensin converting enzyme inhibitor activity by high performance liquid chromatography].

A high performance liquid chromatographic method to determine angiotensin-converting enzyme inhibitor activity in vitro was established by using N-hippuryl-His-Leu tetrahydrate as the reaction substrate and hippuric acid as the reaction product. The chromatographic conditions were as follows: column, ZORBAX SB-C18 (4.6 mm i. d. x 150 mm, 5 microm); column temperature, 25 degrees C; mobile phase, acetonitrile-distilled water (25:75, v/v, both containing 0.05% (v/v) trifluoroacetic acid and 0.1% (v/v) triethylamine); flow rate, 0.5 mL/min; detection wavelength, 228 nm. An excellent linearity over the range of 0.005-1.000 mmol/L (r = 0.9999) was observed. The detection limit was 0.50 micromol/L. The recoveries of hippuric acid ware 99.48%-105.64%, with a relative standard deviation (RSD) of 2.20% (n = 6). It is a simple, precise and reliable assay method for developing antihypertension drugs.