Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Effect of Support Matrix and Crosslinking Agents on Nutritional Properties of Orange Juice during Enzyme Clarification: A Comparative Study.

This study investigated the impact of a support matrix and active group on the support to the nutritional properties of orange juice after juice clarification. Pectinase was immobilized on chitosan and aminated silica supports, activated with genipin or glutaraldehyde, and applied for juice clarification. The effects on various juice properties, including reducing sugars, total soluble solids, vitamin C, and phenolic compounds, juice color, and pH, were evaluated. The results revealed that the immobilization on chitosan activated using genipin resulted in the highest biocatalyst activity (1211.21 U·g-1). The juice treatments using the biocatalysts led to turbidity reduction in the juice (up to 90%), with the highest reductions observed in treatments involving immobilized enzyme on chitosan. Importantly, the enzymatic treatments preserved the natural sugar content, total soluble solids, and pH of the juice. Color differences between treated and raw juice samples were especially relevant for those treated using enzymes, with significant differences in L* and b*, showing loss of yellow vivid color. Analysis of phenolic compounds and vitamin C showed no significant alterations after the enzymatic treatment of the raw juice. According to our results, the clarification of orange juice using immobilized enzymes can be a compromise in turbidity reduction and color reduction to maintain juice quality.

A Pilot Study to Evaluate Genipin in Staphylococcus aureus and Pseudomonas aeruginosa Keratitis Models: Modulation of Pro-Inflammatory Cytokines and Matrix Metalloproteinases.

Infectious keratitis is a vision-threatening microbial infection. The increasing antimicrobial resistance and the fact that severe cases often evolve into corneal perforation necessitate the development of alternative therapeutics for effective medical management. Genipin, a natural crosslinker, was recently shown to exert antimicrobial effects in an ex vivo model of microbial keratitis, highlighting its potential to serve as a novel treatment for infectious keratitis. This study aimed to evaluate the antimicrobial and anti-inflammatory effects of genipin in an in vivo model of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) keratitis. Clinical scores, confocal microscopy, plate count, and histology were carried out to evaluate the severity of keratitis. To assess the effect of genipin on inflammation, the gene expression of pro- and anti-inflammatory factors, including matrix metalloproteinases (MMPs), were evaluated. Genipin treatment alleviated the severity of bacterial keratitis by reducing bacterial load and repressing neutrophil infiltration. The expression of interleukin 1B (IL1B), interleukin 6 (IL6), interleukin 8 (IL8), interleukin 15 (IL15), tumor necrosis factor-α (TNF-α), and interferon γ (IFNγ), as well as MMP2 and MMP9, were significantly reduced in genipin-treated corneas. Genipin promoted corneal proteolysis and host resistance to S. aureus and P. aeruginosa infection by suppressing inflammatory cell infiltration, regulating inflammatory mediators, and downregulating the expression of MMP2 and MMP9.

Aging-related genes related to the prognosis and the immune microenvironment of acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML), one of the common malignancies of the hematologic system, has progressively increased in incidence. Aging is present in both normal tissues and the tumor microenvironment. However, the relationship between senescence and AML prognosis is still not elucidated. METHODS: In this study, RNA sequencing data of AML were obtained from TCGA, and prognostic prediction models were established by LASSO-Cox analysis. Differences in immune infiltration between the different risk groups were calculated using the CIBERSORT and ESTIMATE scoring methods. The KEGG and GO gene enrichment and GSEA enrichment were also used to enrich for differential pathways between the two groups. Subsequently, this study collected bone marrow samples from patients and healthy individuals to verify the differential expression of uncoupling protein 2 (UCP2) in different populations. Genipin, a UCP2 protein inhibitor, was also used to examine its effects on proliferation, cell cycle, and apoptosis in AML cell lines in vitro. RESULTS: It showed that aging-related genes (ARGs) expression was correlated with prognosis. And there was a significant difference in the abundance of immune microenvironment cells between the two groups of patients at high risk and low risk. Subsequently, UCP2 expression was found to be elevated in AML patients. Genipin inhibits UCP2 protein and suppresses the proliferation of AML cell lines in vitro. CONCLUSION: ARGs can be used as a predictor of prognosis in AML patients. Moreover, suppressing UCP2 can reduce the proliferation of AML cell lines, alter their cell cycle, and promote apoptosis in vitro.

Development of a biocomposite based on alginate/gelatin crosslinked with genipin for ß-galactosidase immobilization: Performance and characteristics.

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for ß-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized ß-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.

Fructans with different degrees of polymerization and their performance as carrier matrices of spray dried blue colorant.

Inulin-type fructans with different degrees of polymerization (DPs) were used as wall materials for the blue colorant produced from the crosslinking between genipin and milk proteins. The impact of using fructooligosaccharides (FOS) with DP = 5 and inulins with DP ≥ 10 (GR-In) and DP ≥ 23 (HP-In) on the physical (microstructure, size, water activity, wettability, solubility, water adsorption, glass transition temperature, and color), chemical (free genipin retention and moisture), and technological (colorant power, pH stability, and thermal stability) properties of the powdered blue colorant was examined. Inulins were more efficient carriers as seen from the physical characteristics of the microparticles. FOS and GR-In promoted higher retention of free genipin than HP-In. Additionally, their lower DP influenced the rehydration proprieties as well as the color intensity and colorant power. The DP did not affect the physical stability of the colorant at different pH conditions or at high temperature. Our findings demonstrated that the DP of the fructan exhibited a strong impact on the blue intensity of the samples and also their rehydration capacity.

Genipap (Genipa americana L.) fruit extract as a source of antioxidant and antiproliferative iridoids.

Iridoid blue-based pigments can be found in fruits of genipap (Genipa americana L.). Besides being a potential source of natural blue colorant in the food industry, they have also been associated with pharmacological effects. Therefore, the recovery of iridoids by ultrasound-assisted extraction from both unripe and ripe fruits was analysed by UPLC-DAD-ESI-(-)-QTOF-MS/MS. Nine iridoids were identified from their exact masses and fragmentation pattern, namely geniposidic acid, gardenoside, genipin-1-ß-gentiobioside, geniposide, 6''-O-p-coumaroyl-1-ß-gentiobioside geniposidic acid, 6''-O-p-coumaroylgenipin-gentiobioside, genipin, 6'-O-p-coumaroyl-geniposidic acid and 6'-O-feruloyl-geniposidic acid. Among them, genipin (60.77 mg/g fdw) was found to be the most abundant iridoid in unripe genipap extract, while the ripe genipap extract mainly contained geniposide and geniposidic acid (89.48 and 25.04 mg/g fdw, respectively). It was also observed that the iridoids of the unripe genipap extract are able to scavenger DPPH, ABTS and peroxyl radicals as well as exerting a cytostatic effect against both glioma and breast cancer cell lines. This study provided information about the properties of unripe and ripe genipap extracts which can be used as a reference for further studies focusing on the potential application of G. americana L. in commercial products containing natural blue colorant with functional claims.

Efficient enzyme-assisted extraction of genipin from genipap (Genipa americana L.) and its application as a crosslinker for chitosan gels.

Enzyme-assisted extraction in liquid-liquid two-phase aqueous system was applied for the first time in order to extract genipin from genipap. The effect of different commercial enzymes, their concentrations, and extraction parameters were investigated. Moreover, chitosan gels were prepared, crosslinked with glutaraldehyde or genipin and characterized by their textural and rheological properties. The crosslinked chitosan was used as support for the immobilization of model ß-galactosidases. Among the different commercial enzymes tested for extraction, Celluclast 10% (36 °C and pH 3.7) provided an extraction of 196 mg.g-1 of genipin. Chitosan gels crosslinked with genipin 0.5% showed better textural and similar rheological properties when compared to the chitosan crosslinked with glutaraldehyde 3%. The percentage of lactose hydrolysis by the immobilized K. lactis ß-galactosidase using genipin as a crosslinker was 87%. Thus, the genipin obtained in this work proved to be an excellent alternative to the use of glutaraldehyde in chitosan crosslinking applications.

Extraction of bioactive compounds from genipap (Genipa americana L.) by pressurized ethanol: Iridoids, phenolic content and antioxidant activity.

The search for compounds with functional properties from natural sources has grown in recent years as people have developed healthier habits. Therefore, the aim of this study was to evaluate the extraction of bioactive compounds from various parts of unripe genipap fruit (Genipa americana L.) by using pressurized ethanol to verify which part of the fruit provides the greatest recovery of the iridoids genipin and geniposide. Two process variables were studied: temperature (50 and 80°C) and pressure (2, 12 and 20 bar). The whole fruit and the peel, mesocarp, endocarp, endocarp+seeds and seeds of the fruit were studied. The endocarp presented with the highest recovery of genipin (48.6±0.6mg/g raw material) and the extraction from the mesocarp allowed a greater recovery of geniposide (59±1mg/g raw material). The highest values of total phenolic content were obtained with mesocarp extracts. The endocarp and mesocarp extracts presented the highest antioxidant activity as measured by FRAP and DPPH. These results are promising and support the use of unripe genipap fruit as a source of iridoids and natural antioxidants.

Influence of natural and synthetic crosslinking reagents on the structural and mechanical properties of chitosan-based hybrid hydrogels.

The objective of this work was to correlate the physical and chemical properties of chitosan/poly(vinyl alcohol)/genipin (CS/PVA/GEN) and chitosan/poly(vinyl alcohol)/glutaraldehyde (CS/PVA/GA) hydrogels with their structural and mechanical responses. In addition, their molecular structures were determined and confirmed using FTIR spectroscopy. The results indicated that the hybrid hydrogels crosslinked with genipin showed similar crystallinity, thermal properties, elongation ratio and structural parameters as those crosslinked with glutaraldehyde. However, it was found that the elastic moduli of the two hybrid hydrogels were slightly different: 2.82±0.33MPa and 2.08±0.11MPa for GA and GEN, respectively. Although the hybrid hydrogels crosslinked with GEN presented a lower elastic modulus, the main advantage is that GEN is five to ten thousand times less cytotoxic than GA. This means that the structural and mechanical properties of hybrid hydrogels crosslinked with GEN can easily be tuned and could have potential applications in the tissue engineering, regenerative medicine, food, agriculture and environmental industries.

Structure, morphology and properties of genipin-crosslinked carboxymethylchitosan porous membranes.

Highly porous genipin cross-linked membranes of carboxymethylchitosan exhibiting different crosslinking degree (3%