Optimization of enzymatic production of anti-skin aging biopeptides from white sorghum [Sorghum bicolor (L) Moench] grain.

Recently, kafirins from white sorghum [Sorghum bicolor (L) Moench] grain have shown promise as a source of biopeptides with anti-skin aging effects (anti-inflammatory, antioxidant, and inhibition of photoaging-associated enzymes). This study employed response surface methodology (RSM) to optimize the extraction and enzymatic hydrolysis of kafirins (KAF) for the production of peptides with anti-skin aging properties. The optimization of conditions (reaction time and enzyme/substrate ratio) for liquefaction with α-amylase and hydrolysis of KAF with alcalase was performed using 32 complete factorial designs. Subsequently, ultrafiltered peptide extracts were obtained with molecular weights of 1-3 kDa (KAF-UF3) and lower than 1 kDa (KAF-UF1), which mainly contain hydrophobic amino acids (proline, leucine, isoleucine, phenylalanine, and valine) and peptide fractions with molecular weights of 0.69, 1.14, and 1.87 kDa. Consequently, the peptide extracts protected immortalized human keratinocytes (HaCaT cells) from ultraviolet B radiation (UVB)-induced damage by preventing the decrease and/or restoring the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)]. Furthermore, KAF-UF3 and KAF-UF1 inhibited (20-29%) elastase and collagenase overactivity in UVB-exposed murine fibroblasts (3T3 cells). Thus, KAF-UF3 and KAF-UF1 exhibited behavior similar to that observed with glutathione (GSH), suggesting their potential as functional peptide ingredients in skincare products.

Antioxidant activity of kafirin hydrolysates on UVB irradiated human keratinocyte cells and in silico identification.

BACKGROUND: Ultraviolet B (UVB) causes photoaging of the skin, the appearance of wrinkles, spots, and alteration of the skin barrier. The main cells in the most superficial layer of the skin are the keratinocytes; these cells play an important role in protecting this organ. OBJECTIVE: The present study aimed to investigate the antioxidant activity of the hydrolysates from kafirin to inhibit UVB-induced responses in human keratinocytes cells (HaCaT). METHODS: Kafirin hydrolysates were produced by enzymatic hydrolysis with alcalase. The activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), in the HaCaT cell line in the presence of UVB and the effects of the hydrolysates against the UVB-induced response were evaluated. Furthermore, the peptides that were generated by hydrolysis were identified in silico using the BIOPEP database. RESULTS: Two protein sequences were identified (α-kafirin and the precursor protein of α-kafirin), in the kafirin extract. A degree of hydrolysis of 18.8% was obtained by hydrolyzing the kafirin extract with alcalase. The kafirin hydrolysates avoided the decrease in endogenous antioxidant enzymes such as SOD, CAT, and GPx reducing the oxidative stress generated by UVB. Using the BIOPEP-UWM database, we found 102 peptide sequences, and it has shown that the peptides have a large amount of hydrophobic amino acids such as proline, alanine, and glutamine, and amino acids with high antioxidant capacity. CONCLUSION: These results suggest that the kafirin hydrolysates can be used as antioxidant agents to ameliorate UVB-induced skin keratinocytes cells' response in vitro, providing an alternative against UVB-induced photoaging.

[Antiproliferative, apoptotic, and antimigratory activities of kafirins on cervical cancer-derived cell lines]. / Actividades antiproliferativas, apoptóticas y antimigratorias de las kafirinas en líneas celulares derivadas de cáncer cervical.

Background: Cervical cancer is one of the leading causes of death in women worldwide, both in developed and developing countries. Therefore, effective treatment of cervical cancer with potential anti-tumor drugs is important. However, new treatments inspired by nutritional medicine are needed. Objective: To use the human cervical cancer cell lines HeLa and SiHa to evaluate the antiproliferative, apoptotic, and migratory activity of sorghum (kafirins). Materials and methods: The anticancer effects of the kafirins were examined by counting cells, MTT assays, apoptosis, and migration assays. Results: This investigation showed that sorghum induced growth inhibition of HeLa and SiHa cells at a significant level. The growth inhibition is dose-dependent and irreversible. When HeLa and SiHa cells were treated with sorghum due to the activity of kafirins, morphological changes were observed, which were identified through the formation of apoptopic bodies. And the kafirins at concentrations of 37.5, 75, 150, and 300 µg/mL decreased the migration of HeLa cells and SiHa cells. Conclusion: This paper demonstrates the induction of antiproliferative, apoptotic, and anti-migratory activity in HeLa and SiHa cells by kafirins. Sorghum may be used as a nutraceutical with potential cancer-prevention benefits.

Introducción: el cáncer de cuello uterino es una de las principales causas de muerte en las mujeres de todo el mundo, tanto en los países desarrollados como en los países en desarrollo. Por lo tanto, es importante un tratamiento eficaz de este cáncer con posibles fármacos antitumorales. Sin embargo, se necesitan nuevos tratamientos inspirados en la medicina nutricional. Objetivo: usar las líneas celulares de cáncer cervicouterino humano HeLa y SiHa para evaluar la actividad antiproliferativa, apoptótica y migratoria del sorgo (kafirinas). Material y métodos: los efectos anticancerígenos de las kafirinas se examinaron mediante recuento de células, ensayos de MTT, apoptosis y ensayos de migración. Resultados: la presente investigación demostró que el sorgo induce la inhibición del crecimiento de las células HeLa y SiHa a un nivel significativo. La inhibición del crecimiento es dependiente de la dosis e irreversible. Cuando las células HeLa y SiHa fueron tratadas con sorgo debido a la actividad de las kafirinas, se observaron cambios morfológicos, los cuales se identificaron mediante la formación de cuerpos apoptóticos. Y las kafirinas en concentraciones de 37.5, 75, 150 y 300 µg/mL disminuyeron la migración de las células HeLa y SiHa. Conclusiones: este trabajo demuestra la inducción de actividad antiproliferativa, apoptótica y antimigratoria en las células HeLa y SiHa por parte de las kafirinas. El sorgo puede utilizarse como nutracéutico con potenciales beneficios en la prevención del cáncer.

Delayed Senescence and Marketability Index Preservation of Blackberry Fruit by Preharvest Application of Chitosan and Salicylic Acid.

Blackberry fruits are appreciated as a source of nutrients and compounds related to benefit human health. However, they are highly perishable and very susceptible to decay factors. Current methods to improve and maintain blackberry quality are limited in use because of the fruit's fragile physical properties. Regarding these properties, it has been reported that the activities of certain enzymes are linked to senescence and fruit softening processes. This study was aimed to assess the effect of salicylic acid (SA) and chitosan (COS) as preharvest treatments on the physiology related to improving fruit conservation and preserving the marketability index of blackberry fruit. The preharvest treatments were foliar sprayed on blackberry plants at different concentrations. The activities of enzymes superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia-lyase (PAL), and polygalacturonase (PG) were measured. Total soluble solids (TSS), titratable acidity (TA), TSS/TA ratio, and marketability index (MI) were analyzed after 144 h of storage. The application of 3 mM of SA and 0.25% of COS treatments preserved the MI of blackberries by reducing leakage, red drupelet reversion (RDR), and mycelium presence in the fruit. SA application increased SOD, CAT, and PAL activities. Our results also showed that SA and COS preharvest treatments modified the activity of the cell wall degrading enzyme PG, which might play a role in improving the shelf life and resistance to decay factors of blackberry fruit without any significant effects on physicochemical properties like TSS, TA, and the TSS/TA ratio.

Optimization of Sorghum Kafirin Extraction Conditions and Identification of Potential Bioactive Peptides.

The interest in extracting kafirins (KAF), the main storage protein from sorghum grain has recently increased due to its gluten-free content and the significant scientific evidence showing the health benefits of the bioactive peptides from cereal grains in human diets. The objectives were to obtain the highest percentage of KAF extraction using amyloglucosidase as pretreatment to increase the extraction yield and predict the bioactive peptides in the KAF. In this study, pretreatments with amyloglucosidase increased the extraction yield of KAF compared with extraction methods using only ethanol and sodium metabisulfite. Two protein fragment sequences were identified from KAF extract and were evaluated for potential bioactive peptide using the BIOPEP-UWM database, which suggest that KAF proteins from white sorghum may be considered as good precursors of dipeptidyl peptidase-inhibitor, angiotensin-converting enzyme inhibitor, antioxidant and hypotensive peptides following chymotrypsin, thermolysin, and subtilisin and their combination. Average scores aligned using PeptideRanker confirmed KAF proteins' potential sources of bioactive peptides with over 5 peptides scored over 0.8. In addition, 31 unexplored peptide sequences that could have biological activity were identified. Our results suggest that KAF can be used in the peptide productions with potential biological activity and beyond.

Activation of the phenylpropanoid biosynthesis pathway reveals a novel action mechanism of the elicitor effect of chitosan on avocado fruit epicarp.

Secondary metabolites play an important role in the avocado fruit defense system. Phenolic compounds are the main biosynthesized metabolites of this system response. Our objective in this investigation was to evaluate the induction of specific metabolic pathways using chitosan as an elicitor. Extracts obtained from avocado in intermediate and consumption maturity stages treated with chitosan exhibited an increase in antifungal activity, which caused inhibition of mycelial growth and a decrease in sporulation as well as spore germination of Colletotrichum gloeosporioides. Additionally, RNA from epicarp of the fruits treated and untreated with chitosan was obtained in order to evaluate the expression of genes related to phenylpropanoids and the antifungal compound 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene biosynthesis. An increased in gene expression of genes that participates in the phenylpropanoids route was observed during the stage of physiological fruit maturity, others genes such as Flavonol synthase (Fls), increased only in samples obtained from fruit treated with chitosan at consumption maturity. Our results reveal a new molecular mechanism where chitosan induces a specific accumulation of phenylpropanoids and antifungal diene; this partially explains avocado's resistance against fungal pathogens. Finally, we discuss the molecular connections between chitosan induction and gene expression to explain the biological events that orchestrate the resistance pathways in fruits.

Identification and characterization of a new Bacillus atrophaeus strain B5 as biocontrol agent of postharvest anthracnose disease in soursop (Annona muricata) and avocado (Persea americana).

Anthracnose is a fungal disease caused by Colletotrichum species that is detrimental to numerous fruit, including soursop and avocado. The use of fungicides to maintain the high quality of fruit creates a potential health risk. One alternative to this problem is the biological control, which has been applied successfully during postharvest. The Bacillus species are one of the most studied biological agents against postharvest pathogens because accomplish their biocontrol performance by producing a variety of metabolites. In this study, we evaluated the activity of metabolites contained in the cell free supernatant, obtained from Bacillus strain B5 culture, against micelial growth and spore germination of two virulent strains of C. gloeosporioides isolated from soursop and avocado. On the basis of 16S rDNA gene sequence analysis, this strain was identified as Bacillus atrophaeus. A preventive treatment using cell free supernatant, reduced severity and incidence of anthracnose disease on harvested soursop and avocado fruit. B. atrophaeus strain B5 harbors genes involved in the production of antibiotics such as surfactin, bacillomycin and iturin, which could be contributing to the efficiency of the preventive treatment during postharvest. The antagonistic role of metabolites contained in the cell free supernatant against anthracnose disease, provide a new approach by which to attack this problem and can help reduce the use of chemical pesticides, environmental pollution, leading to the safer fruit preservation.