Comparative Study on the Mechanism of Macrophage Activation Induced by Polysaccharides from Fresh and Dried Longan.

Longan (Dimcarpus longan Lour.) is a kind of traditional fruit used as a medicine and a food. Fresh longan is primarily consumed as a fruit, whereas dried longan is commonly employed for medicinal purposes. The differences in the immunomodulatory activities and mechanisms of polysaccharides between dried and fresh longan remain unclear. The present study comparatively analyzed the mechanisms of macrophage activation induced by polysaccharides from dried (LPG) and fresh longan (LPX). The results revealed that LPG and LPX differentially promoted macrophage phagocytosis and the secretion of NO, TNF-α, and IL-6. RNA-seq analysis revealed that LPG and LPX differentially affected gene expression in macrophages. The LPG treatment identified Tnf and chemokine-related genes as core genes, while myd88 and interferon-related genes were the core genes affected by LPX. A comprehensive analysis of the differentially expressed genes showed that LPG initiated macrophage activation primarily through the TLR2/4-mediated TRAM/TRAF6 and CLR-mediated Src/Raf1 NF-κB signaling pathways. LPX initiated macrophage activation predominantly via the CLR-mediated Bcl10/MALT1 and NLR-mediated Rip2/TAK1 MAPK and NF-κB signaling pathways. Interestingly, the non-classical NF-κB signaling pathway was activated by polysaccharides in both dried and fresh longan to elicit a slow, mild immune response. LPG tends to promote immune cell migration to engage in the immune response, while LPX facilitates antigen presentation to promote T cell activation. These findings contribute insights into the mechanisms underlying the differences in bioactivity between dried and fresh longan and their potential applications in immune-enhancing strategies and functional-food development.

PeposX-Exhaust: A lightweight and efficient tool for identification of short peptides.

Short peptides have become the focus of recent research due to their variable bioactivities, good digestibility and wide existences in food-derived protein hydrolysates. However, due to the high complexity of the samples, identifying short peptides still remains a challenge. In this work, a tool, named PeposX-Exhaust, was developed for short peptide identification. Through validation with known peptides, PeposX-Exhaust identified all the submitted spectra and the accuracy rate reached 75.36%, and the adjusted accuracy rate further reached 98.55% when with top 5 candidates considered. Compared with other tools, the accuracy rate by PeposX-Exhaust was at least 70% higher than two database-search tools and 15% higher than the other two de novo-sequencing tools, respectively. For further application, the numbers of short peptides identified from soybean, walnut, collagen and bonito protein hydrolysates reached 1145, 628, 746 and 681, respectively. This fully demonstrated the superiority of the tool in short peptide identification.

Comparative peptidomics analysis in the discovery of umami peptides from Chinese Douchi.

Douchi is a kind of traditional Chinese fermented soybean product with outstanding umami taste. Besides the umami amino acids in Douchi, peptides were also considered as an important contributor for the umami taste of Douchi. Peptides with molecular weight below 0.66 kDa accounted for more than 50 % in all samples except for TongChuan Douchi, and a total of 421 peptides were identified from the ten kinds of Douchi samples by using LC-MS/MS. Combined with sensory evaluation results, 19 peptides containing Glu, Asp or known umami peptide sequences were chosen as potential umami peptides via PLS-DA and RDA analysis. Among them, 17 soluble peptides exhibited obvious umami taste and the threshold of 7 peptides were lower than MSG solution. Especially, the VD was detected with a minimum umami taste threshold at 0.16 mg/mL. The results indicated that the umami peptides might be the important components affecting the umami taste of Douchi.

Screening of novel bovine-elastin-derived peptides with elastase inhibition and photoprotective potential: a combined in silico and in vitro study.

BACKGROUND: The demand for food-based anti-photoaging products is surging because of the rising recognition of health and beauty, as well as enhanced comprehension of the detrimental impact of ultraviolet (UV) radiation. This study aimed to investigate the potential of bioactive peptides derived from bovine elastin, specifically focusing on identifying novel elastase inhibitory peptides and assessing their photoprotective properties using bioinformatics techniques. RESULTS: A total of 48 bioactive peptides were screened in bovine elastin hydrolysate (EH) utilizing Peptide Ranker analysis. Three novel elastase inhibitory peptides, GAGQPFPI, FFPGAG and FPGIG (in descending order of activity), exhibited potent inhibitory effects on elastase in vitro, surpassing the inhibitory effect of EH by a factor of 1-2 and reaching significantly lower concentrations (8-15 times lower) than EH. The cumulative inhibitory effect of GAGQPFPI, FFPGAG, and FPGIG reached 91.5%. Further analysis revealed that FFPGAG and FPGIG exhibited mixed inhibition, whereas GAGQPFPI displayed non-competitive inhibition. Molecular simulations showed that these peptides interacted effectively with the elastase active site through hydrogen bonding and hydrophobic interactions. Furthermore, GAGQPFPI, FFPGAG, and FPGIG demonstrated high stability in gastrointestinal digestion, demonstrated transcellular permeability across Caco-2 cell monolayers, and exhibited remarkable photoprotective properties against UVB-irradiated HaCaT cells. CONCLUSION: GAGQPFPI showed the most promising potential as a functional food with photoprotective effects against UVB damage and inhibitory properties against elastase. © 2023 Society of Chemical Industry.

Enhancing Kokumi Sensation and Reducing Bitterness in Acid-Hydrolyzed Vegetable Proteins through Lactate and Thermal Processing.

Previous studies have demonstrated that thermal processing in the presence of lactate and amino acids can produce taste-active N-lactoyl amino acids. This study aimed to investigate the impact of lactate and thermal processing on the sensory characteristics of acid-hydrolyzed vegetable proteins (aHVP). The results showed that the processed aHVP exhibited enhanced kokumi, a milder umami taste, and reduced bitterness on treatment with 1% lactate at 110 °C for 3 h or 3% lactate at 120 °C for 2 h compared to the unprocessed samples. Partial or orthogonal least-squares discriminant analysis and variable importance in projection (VIP) analyses revealed the significant contributions of N-,l-Lac-l-hydrophobic AAs [-Met, -Ile, -Leu, -Val, and -Phe (VIP > 1.2)] to the observed differences between the processed and unprocessed samples. Electronic tongue analysis confirmed the sensory findings and indicated a decrease in the aftertaste of bitterness in the processed samples. Furthermore, the study identified the sensory characteristics of N-l-Lac-l-Met, -Ile, and -Leu, highlighting their potential to enhance salty, umami, and kokumi perception in simulated broth. Furthermore, the study incorporated the addition of bitter amino acids (Val, Ile, Leu, Tyr, Phe, Lys, His, and Arg) and the aforementioned N-l-Lac-l-AAs to aHVP, providing further evidence for their contributions to bitterness and aftertaste-B as well as the kokumi differences, respectively. This study provides valuable insights into the sensory effects of lactate and thermal processing on aHVP, facilitating the development of improved taste-enhancing strategies.

Effects of simulated digestion on the structural characteristics and dendritic cell activation of longan polysaccharides.

An active polysaccharide (LP) from longan was purified and characterized. LP consisted of galactose and glucose in a molar ratio of 1.5: 98.5, with a molecular weight of 4.67 × 107 g/mol. The main backbone of LP was T-α-D-Glcp-[(1 â†’ 6)-α-D-Glcp-(1 â†’ 6)-α-D-Glcp]n. After simulated gastrointestinal digestion, the molecular weight distribution, monosaccharide composition, and major glycosidic bonds of LP were not significantly changed. LP and digested LP (DLP) reduced phagocytosis and promoted IL-10 and IL-12 secretion of dendritic cells. In addition, the effects of LP and DLP on activating dendritic cells showed no significant difference. This study helps to illuminate the potential mode of immunomodulatory action of longan polysaccharides in vivo.

Efficient extraction, excellent activity, and microencapsulation of flavonoids from Moringa oleifera leaves extracted by deep eutectic solvent.

A deep eutectic solvent (choline chloride (ChCl)-urea) was chosen to extract flavonoids from Moringa oleifera leaves (FMOL), the condition of extraction was tailor-made, under the optimal extraction conditions (material-to-liquid ratio of 1:60 g/mL, extraction time of 80 min, extraction temperature of 80 °C), the highest extraction efficiency reached 63.2 ± 0.3 mg R/g DW, and nine flavonoids were identified. Then, the biological activities including antioxidant activities, antibacterial activities, and anti-tumor activities were systematically studied. FMOL was superior to positive drugs in terms of antioxidant activity. As to DPPH investigation, the IC50 of FMOL and Vc were 64.1 ± 0.7 and 176.1 ± 2.0 µg/mL; for the ABTS, the IC50 of FMOL and Vc were 9.5 ± 0.3 and 38.2 ± 1.2 µg/mL, the FRAP value of FMOL and Vc were 15.5 ± 0.6 and 10.2 ± 0.4 mg TE/g, and ORAC value of FMOL and Vc were 4687.2 ± 102.8 and 3881.6 ± 98.6 µmol TE/g. The bacteriostatic (MICs were ≤ 1.25 mg/mL) activities of FMOL were much better than propyl p-hydroxybenzoate. Meanwhile, FMOL had comparable inhibitory activity with genistein on tumor cells, IC50 was 307.8 µg/mL, and could effectively induce apoptosis in HCT116. Microcapsules were prepared with xylose-modified soybean protein isolate and gelatin as wall materials; after that, the intestinal release of modified FMOL microcapsules was 86 times of free FMOL. Therefore, this study confirmed that FMOL extracted with ChCl/urea has rich bioactive components, and microencapsulated FMOL has potential application in food industry. Supplementary Information: The online version contains supplementary material available at 10.1007/s13399-023-03877-8.

Discovery of N-l-Lactoyl-l-Trp as a Bitterness Masker via Structure-Based Virtual Screening and a Sensory Approach.

N-Lactoyl-amino acid derivatives (N-Lac-AAs) are of increasing interest as potential taste-active compounds. The complexity and diversity of N-Lac-AAs pose a significant challenge to the effective discovery of taste-active N-Lac-AAs. Therefore, a structure-based virtual screening was used to identify taste-active N-Lac-AAs. Virtual screening results showed that N-lactoyl-hydrophobic amino acids had a higher affinity for taste receptors, specifically N-l-Lac-l-Trp. And then, N-l-Lac-l-Trp was synthesized in yields of 22.3% by enzymatic synthesis in the presence of l-lactate and l-Trp, and its chemical structure was confirmed by MS/MS and one-dimensional (1D) and two-dimensional (2D) NMR. Sensory evaluation revealed that N-l-Lac-l-Trp had a significant taste-masking effect on quinine, d-salicin, caffeine, and l-Trp, particularly l-Trp and caffeine. N-l-Lac-l-Trp had a better masking effect on the higher concentration of bitter compounds. It reduced the bitterness of caffeine (500 mg/L) and l-Trp (1000 mg/L) by approximately 20 and 26%, respectively. The result of the ligand-receptor interaction and a quantum mechanical analysis showed that N-l-Lac-l-Trp increased the binding affinity to the bitter receptor mainly through hydrogen bonding and lowering the electrostatic potential.

A fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry and its application in food-derived protein hydrolysates.

Food-derived bioactive peptides have many outstanding features like high safety, easy absorption, etc. However, explorations of the peptides are suffering from the limited knowledge of sample composition and low efficiency of separation techniques. In this work, a fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry (2DLC-MS) was designed and constructed in-house. For chromatographic system optimization, the effects of column pairs and fraction transfer volumes on separation performance were studied. The pair of Protein BEH SEC and HSS T3 columns was found of high orthogonality. The peak capacity detected by the optimized 2DLC reached 1165 (for corn protein hydrolysates), indicating high resolving power. Moreover, the number of peptides identified from corn, soybean and casein protein hydrolysates reached as high as 8330, 8925 and 7215, respectively, demonstrating the high potential of the system. This would help reveal the peptide composition and facilitate the research on exploring bioactive peptides from food-derived protein hydrolysates.

Glycyrrhetinic acid proliposomes mediated by mannosylated ligand: Preparation, physicochemical characterization, environmental stability and bioactivity evaluation.

Glycyrrhetinic acid is a bioactive compound extracted from licorice that exhibits inhibition effect on various cancers. However, its hydrophobicity results in low bioavailability that limits application. We aim to overcome this barrier, the present research was performed to prepare glycyrrhetinic acid proliposomes mediated mannosylated ligand (mannose-diester lauric diacid-cholesterol, MDC) and to evaluate its physicochemical characterizations, environmental stability and bioactivity. In preliminary optimization studies of glycyrrhetinic acid proliposomes mediated MDC (MDC-GA-PL), four optimum operating parameters, cryoprotectant of glucose and mannitol, the mixed cryoprotectant ratio (glucose/mannitol) of 1:1, a cryoprotectant/egg phosphatidylcholine mass ratio of 10/1, and -60 â„ƒ pre-freezing temperature, were obtained after investigation. Under the optimum lyophilization conditions, MDC-GA-PL was freeze-dried and reconstituted proliposomes were characterized. These proliposomes showed that MDC-GA-PL were well-dispersible spherical particles with an average particle size of 120.80 nm, a polydispersity index about 0.095, a zeta potential of -33.15 mV, encapsulation efficiency of 85.9% and drug loading of 6.38%. In vitro drug release study showed that glycyrrhetinic acid release of MDC-GA-PL conforms to the Higuchi release model. In addition, these proliposomes were stable during six months at 4 â„ƒ. Moreover, acute toxicity assay revealed no substantial safety concern for MDC-GA-PL. Finally, in vitro bioactivity of proliposomes was evaluated. Cytotoxicity effect and apoptosis efficiency of MDC-GA-PL by HepG2 cells was significantly higher than that of glycyrrhetinic acid proliposomes without MDC, demonstrating that MDC has a desirable effect on liver target. Overall, we have reason to believe that MDC-GA-PL would be a promising target delivery to improve therapeutic against hepatic diseases.

Generation of a homozygous ARHGAP11B knockout hiPSC line by CRISPR/Cas9 system.

The human specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal neocortex and plays a key role in the evolutionary expansion of the neocortex. Here, we generated a homozygous ARHGAP11B knockout human induced pluripotent stem cell (hiPSC) line through CRISPR/Cas9 gene editing system. ARHGAP11B deficient cell line maintained a normal karyotype (46, XX), expressed pluripotency markers, and showed the capability to spontaneously differentiate into all three germ layers in vivo. The ARHGAP11B knockout cell line can provide a new cell model for studying the evolution of human neocortex.

PAYCS Alleviates Scopolamine-Induced Memory Deficits in Mice by Reducing Oxidative and Inflammatory Stress and Modulation of Gut Microbiota-Fecal Metabolites-Brain Neurotransmitter Axis.

The bioactive peptide PAYCS (Pro-Ala-Tyr-Cys-Ser) identified from anchovy hydrolysates has been reported to be positive in memory alleviation. The gut microbiota-brain axis plays a vital role in brain functions, which could be affected by nutritional supplementation. Herein, we found that PAYCS at different concentrations (PAYCS-L and PAYCS-H) showed various improving effects in behavioral tests and alleviation effects on oxidative as well as inflammatory stress in the scopolamine-induced AD mouse model. The 16S rRNA results illustrated that PAYCS-L altered the ratio of Bacteroidetes/Firmicutes and PAYCS treatment elevated the relative abundance of Cacteroidaceae and Prevotellaceae. Notably, administration of PAYCS significantly upregulated memory-related metabolites and neurotransmitters. Overall, PAYCS-L reversed memory deficits of amnesiac mice partially via the modulation of gut microbiota-metabolites-brain neurotransmitter axis. For PAYCS-H, functions might be involved in the reversal of oxidative and inflammatory impairments in the liver and serum, which was also associated with the changed intestinal microbiota and fecal metabolites.

A Novel Polysaccharide Isolated From Fresh Longan (Dimocarpus longan Lour.) Activates Macrophage via TLR2/4-Mediated PI3/AKT and MyD88/TRAF6 Pathways.

A novel immunomodulatory polysaccharide (LP4) with a molecular weight 6.31 × 104 g/mol was purified from fresh longan pulp. It was composed of mannose, glucose, glucuronic acid, galactose, xylose, arabinose, galacturonic acid, fucose, and rhamnose in a molar percentage of 36:31:10:7:4:4:3:2:2, and mainly linked by (1→6)-ß-Man, (1→4)-ß-Glc and (1→6)-α-Glc. LP4 can obviously enhance the phagocytosis of macrophages and promote the proliferation of lymphocytes. After treating macrophages with LP4 (12.5-50 µg/ml), the production of IL-1ß and TNF-α was significantly increased. These increases of cytokines were suppressed when the TLR2/TLR4 receptors were inhibited by anti-TLR2 and/or anti-TLR4 antibodies. Moreover, the mRNA expression of INOS, AKT, PI3K, TRAF6 and MyD88 was significantly suppressed by TLR2/TLR4 antibodies. These results indicated that LP4 induced macrophage activation mainly via the TLR2 and TLR4-induced PI3K/AKT and MyD88/TRAF6 pathways.

An improved peak clustering algorithm for comprehensive two-dimensional liquid chromatography data analysis.

In this work, an improved algorithm was developed for two-dimensional (2D) peak detection in complex two-dimensional liquid chromatography (LC×LC) data sets. In the first step, conventional one-dimensional peak detection was performed. In the second step, retention time, bidirectional overlap and unimodality criteria were applied to decide which of the individual peaks should be merged. To improve the peak detection with LC×LC analysis using shifting second dimension (2D) gradients, the variable thresholds, which permitted different thresholds for candidate peaks at different first dimension (1D) retention times, were employed for examination of the 2D retention time differences. Furthermore, the bidirectional overlap criterion performed at specified height was recommended to improve detection for tailing peaks. The developed algorithm was further tested on data sets from different LC×LC analyses of a complex peptide mixture, and then quantitatively evaluated by comparison between the results by the algorithm and mass analysis. Evidently improved performance with an accuracy rate over 60% was obtained by the algorithm, even for peak detection with LC×LC analysis under relatively low 1D sampling frequency or shifting 2D gradients. This would help to improve LC×LC quantitative analysis and performance assessment.

Stop-flow reversed phase liquid chromatography × size-exclusion chromatography for separation of peptides.

Size-exclusion chromatography (SEC) with wide application in peptide analysis presents challenges in determination of molecular weight distribution due to the relatively low resolution. In this study, a stop-flow reversed phase liquid chromatography (RPLC) × SEC system was constructed, aiming at improving the peptide separation in SEC. As the chromatographic dispersion during stop-flow operation might contribute to the band broadening in the first dimension (1st D) RPLC, the effects of different stop-flow operational parameters on the additional band broadening were quantitatively evaluated. Unlike analytes of large molecular size or long retention time with low effective diffusion coefficient (Deff), additional band broadening was evidently observed for analytes of small molecular size and short retention time (high Deff). Therefore, optimal flow rate, low column temperature and short stop-flow time were suggested for analyzing small molecules of short retention time. The established stop-flow two-dimensional liquid chromatography (2D-LC) was further tested on protein hydrolysates. The resolution was evidently improved for both heart-cutting and comprehensive 2D-LC analysis (despite additional band broadening in RPLC). Compared with heart-cutting analysis with higher 1st D resolution for selective fractions, comprehensive analysis could provide more complete information about the molecular weight distribution of the eluting solutes along RPLC.

Effect of walnut protein hydrolysate on scopolamine-induced learning and memory deficits in mice.

A walnut protein hydrolysate (WPH) was prepared by using a mixture of pancreatin and viscozyme L from industrially available defatted walnut meal. The antioxidant effects of WPH were confirmed and quantified by reducing power, oxygen radical absorbance capacity, hydroxyl radical radical-scavenging activity and ABTS+· radical-scavenging activity assays. The protective effects of WPH on scopolamine-induced learning and memory deficits in mice were also evaluated based on in vivo behavioral tests. Results showed that WPH administration would lead to significantly decreased latencies while increased crossing times and target times in the spatial probe test, and increased escape latency and decreased error times in the step-down avoidance test for the scopolamine-induced dementia mice. Biochemical results indicated that the ameliorative effects of WPH on scopolamine-induced dementia mice could be attributed to the significantly increased amount of acetylcholine receptors. Therefore, WPH may be a potential therapeutic agent against Alzheimer's disease.

Additional band broadening of peptides in the first size-exclusion chromatographic dimension of an automated stop-flow two-dimensional high performance liquid chromatography.

The need to improve the peak capacity of liquid chromatography motivates the development of two-dimensional analysis systems. This paper presented a fully automated stop-flow two-dimensional liquid chromatography system with size exclusion chromatography followed by reversed phase liquid chromatography (SEC×RPLC) to efficiently separate peptides. The effects of different stop-flow operational parameters (stop-flow time, peak parking position, number of stop-flow periods and column temperature) on band broadening in the first dimension (1st D) SEC column were quantitatively evaluated by using commercial small proteins and peptides. Results showed that the effects of peak parking position and the number of stop-flow periods on band broadening were relatively small. Unlike stop-flow analysis of large molecules with a long running time, additional band broadening was evidently observed for small molecule analytes due to the relatively high effective diffusion coefficient (Deff). Therefore, shorter analysis time and lower 1st D column temperature were suggested for analyzing small molecules. The stop-flow two-dimensional liquid chromatography (2D-LC) system was further tested on peanut peptides and an evidently improved resolution was observed for both stop-flow heart-cutting and comprehensive 2D-LC analysis (in spite of additional band broadening in SEC). The stop-flow SEC×RPLC, especially heart-cutting analysis with shorter analysis time and higher 1st D resolution for selected fractions, offers a promising approach for efficient analysis of complex samples.