Inactivation of guanylate kinase in Bacillus sp. TL7-3 cultivated under an optimized ratio of carbon and nitrogen sources influenced GTP regeneration capability and sporulation.

Bacillus sp. TL7-3 has potential as a dietary supplement to promote human and animal health. It produces spores that can survive in harsh environments. Thus, when supplemented with nutrients, these spores can withstand the acidic pH of the stomach and resume vegetative development in the gut when exposed to growth-promoting conditions. Spores are formed as a cellular defense mechanism when a culture experiences stress and process optimization to achieve high spore production in a typical batch process remains challenging. Existing literature on the manipulation of gene expression and enzyme activity during batch cultivation is limited. Studies on the growth patterns, morphological changes, and relevant gene expression have aided in enhancing spore production. The present study used the response surface methodology for medium optimization. The model suggested that yeast extract and NH4Cl were significant factors controlling spore production. A comparison between the high weight ratio of carbon and nitrogen (C:N) substrates (8.57:1) in the optimized and basal media (0.52:1) showed an 8.76-fold increase in the final spore concentration. The expression of major genes, including codY, spo0A, kinA, and spo0F, involved in the sporulation was compared when cultivating Bacillus sp. TL7-3 in media with varying C:N ratios. At high C:N ratios, spo0A, kinA, and spo0F were upregulated, whereas codY was downregulated. This led to decreased guanylate kinase activity, resulting in a low guanosine triphosphate concentration and inactivation of CodY, thereby reducing the repression of spo0A and CodY-repressed genes and stimulating sporulation.

Association between organic nitrogen substrates and the optical purity of D-lactic acid during the fermentation by Sporolactobacillus terrae SBT-1.

The development of biotechnological lactic acid production has attracted attention to the potential production of an optically pure isomer of lactic acid, although the relationship between fermentation and the biosynthesis of highly optically pure D-lactic acid remains poorly understood. Sporolactobacillus terrae SBT-1 is an excellent D-lactic acid producer that depends on cultivation conditions. Herein, three enzymes responsible for synthesizing optically pure D-lactic acid, including D-lactate dehydrogenase (D-LDH; encoded by ldhDs), L-lactate dehydrogenase (L-LDH; encoded by ldhLs), and lactate racemase (Lar; encoded by larA), were quantified under different organic nitrogen sources and concentration to study the relationship between fermentation conditions and synthesis pathway of optically pure lactic acid. Different organic nitrogen sources and concentrations significantly affected the quantity and quality of D-lactic acid produced by strain SBT-1 as well as the synthetic optically pure lactic acid pathway. Yeast extract is a preferred organic nitrogen source for achieving high catalytic efficiency of D-lactate dehydrogenase and increasing the transcription level of ldhA2, indicating that this enzyme plays a major role in D-lactic acid formation in S. terrae SBT-1. Furthermore, lactate racemization activity could be regulated by the presence of D-lactic acid. The results of this study suggest that specific nutrient requirements are necessary to achieve a stable and highly productive fermentation process for the D-lactic acid of an individual strain.

Lemon basil seed-derived peptide: Hydrolysis, purification, and its role as a pancreatic lipase inhibitor that reduces adipogenesis by downregulating SREBP-1c and PPAR-γ in 3T3-L1 adipocytes.

The purpose of this study is to assess the bioactive peptides derived from the defatted lemon basil seeds hydrolysate (DLSH) for their ability to inhibit pancreatic lipase, decrease intracellular lipid accumulation, and reduce adipogenesis. Response surface methodology (RSM) was employed to optimize trypsin hydrolysis conditions for maximizing lipase inhibitory activity (LI). A hydrolysis time of 387.06 min, a temperature of 49.03°C, and an enzyme concentration of 1.61% w/v, resulted in the highest LI with an IC50 of 368.07 µg/mL. The ultrafiltration of the protein hydrolysate revealed that the fraction below 0.65kDa exhibited the greatest LI potential. Further purification via RP-HPLC identified the Gly-Arg-Ser-Pro-Asp-Thr-His-Ser-Gly (GRSPDTHSG) peptide in the HPLC fraction F1 using mass spectrometry. The peptide was synthesized and demonstrated LI with an IC50 of 0.255 mM through a non-competitive mechanism, with a constant (Ki) of 0.61 mM. Docking studies revealed its binding site with the pancreatic lipase-colipase complex. Additionally, GRSPDTHSG inhibited lipid accumulation in 3T3-L1 cells in a dose-dependent manner without cytotoxic effects. Western blot analysis indicated downregulation of PPAR-γ and SREBP-1c levels under GRSPDTHSG treatment, while an increase in AMPK-α phosphorylation was observed, suggesting a role in regulating cellular lipid metabolism. Overall, GRSPDTHSG demonstrates potential in attenuating lipid absorption and adipogenesis, suggesting a prospective application in functional foods and nutraceuticals.

Isolation, screening, and characterization of the newly isolated osmotolerant yeast Wickerhamomyces anomalus BKK11-4 for the coproduction of glycerol and arabitol.

This study explored the isolation and screening of an osmotolerant yeast, Wickerhamomyces anomalus BKK11-4, which is proficient in utilizing renewable feedstocks for sugar alcohol production. In batch fermentation with high initial glucose concentrations, W. anomalus BKK11-4 exhibited notable production of glycerol and arabitol. The results of the medium optimization experiments revealed that trace elements, such as H3BO3, CuSO4, FeCl3, MnSO4, KI, H4MoNa2O4, and ZnSO4, did not increase glucose consumption or sugar alcohol production but substantially increased cell biomass. Osmotic stress, which was manipulated by varying initial glucose concentrations, influenced metabolic outcomes. Elevated glucose levels promoted glycerol and arabitol production while decreasing citric acid production. Agitation rates significantly impacted the kinetics, enhancing glucose utilization and metabolite production rates, particularly for glycerol, arabitol, and citric acid. The operational pH dictated the distribution of the end metabolites, with glycerol production slightly reduced at pH 6, while arabitol production remained unaffected. Citric acid production was observed at pH 6 and 7, and acetic acid production was observed at pH 7. Metabolomic analysis using GC/MS identified 29 metabolites, emphasizing the abundance of sugar/sugar alcohols. Heatmaps were generated to depict the variations in metabolite levels under different osmotic stress conditions, highlighting the intricate metabolic dynamics occurring post-glucose uptake, affecting pathways such as the pentose phosphate pathway and glycerolipid metabolism. These insights contribute to the optimization of W. anomalus BKK11-4 as a whole-cell factory for desirable products, demonstrating its potential applicability in sustainable sugar alcohol production from renewable feedstocks.

Sulfated polysaccharides from Caulerpa lentillifera: Optimizing the process of extraction, structural characteristics, antioxidant capabilities, and anti-glycation properties.

The polysaccharides found in Caulerpa lentillifera (sea grape algae) are potentially an important bioactive resource. This study makes use of RSM (response surface methodology) to determine the optimal conditions for the extraction of valuable SGP (sea grape polysaccharides). The findings indicated that a water/raw material ratio of 10:1 mL/g, temperature of 90 °C, and extraction time of 45 min would maximize the yield, with experimentation achieving a yield of 21.576 %. After undergoing purification through DEAE-52 cellulose and Sephacryl S-100 column chromatography, three distinct fractions were obtained, namely SGP11, SGP21, and SGP31, each possessing average molecular weights of 38.24 kDa, 30.13 kDa, and 30.65 kDa, respectively. Following characterization, the fractions were shown to comprise glucose, galacturonic acid, xylose, and mannose, while the sulfate content was in the range of 12.2-21.8 %. Using Fourier transform infrared spectroscopy (FT-IR) it was possible to confirm with absolute certainty the sulfate polysaccharide attributes of SGP11, SGP21, and SGP31. NMR (nuclear magnetic resonance) findings made it clear that SGP11 exhibited α-glycosidic configurations, while the configurations of SGP21 and SGP31 were instead ß-glycosidic. The in vitro antioxidant assays which were conducted revealed that each of the fractions was able to demonstrate detectable scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations. All fractions were also found to exhibit the capacity to scavenge NO radicals in a dose-dependent manner. SGP11, SGP21, and SGP31 were also able to display cellular antioxidant activity (CAA) against the human adenocarcinoma colon (Caco-2) cell line when oxidative damage was induced. The concentration levels were found to govern the extent of such activity. Moreover, purified SGP were found to exert strong inhibitory effects upon glycation, with the responses dependent upon dosage, thus confirming the potential for SGP to find a role as a natural resource for the production of polysaccharide-based antioxidant drugs, or products to promote improved health.

Corrigendum: Microalgae-based wastewater treatment for developing economic and environmental sustainability: Current status and future prospects.

[This corrects the article DOI: 10.3389/fbioe.2022.904046.].

Investigating the cellular antioxidant and anti-inflammatory effects of the novel peptides in lingzhi mushrooms.

The lingzhi mushroom (Ganoderma lucidum) is well known for its medicinal properties and has long played a role in traditional oriental medicine due to its health-giving benefits and potential to extend life expectancy. The mushroom contains a number of highly bioactive compounds and can also act as an excellent source of protein. This research investigated the peptides obtained from the protein hydrolysates of lingzhi mushrooms to assess their free radical scavenging abilities. These peptides were acquired via different proteases (Alcalase, Neutrase, papain, and pepsin-pancreatin) and were tested at a range of different concentrations (1.0%, 2.5%, and 5.0% w/v). The highest levels of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging activities were presented by lingzhi mushroom hydrolysate using 2.5% (w/v) pepsin-pancreatin after 6 h of digestion. The hydrolysate was then fractionated using 10, 5, 3, and 0.65 kDa molecular weight cut-off membranes. The results showed that the MW 0.65 kDa fraction had the highest level of free radical scavenging activity. Further analysis of this MW 0.65 kDa fraction began with another RP-HPLC fractionation technique to obtain three further sub-fractions. De novo peptide sequencing using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) was chosen as the optimum method for studying the F3 sub-fraction. DRVSIYGWG and ALLSISSF were discovered as new peptides with different antioxidant properties. Adenocarcinoma colon (Caco-2) cells showed the antioxidant action of these synthesized peptides. This activity was linked to peptide concentration. The peptides and their pure synthetic counterparts were found to reduce NO generation by RAW 264.7 macrophages without causing cytotoxicity. The results of gene expression reveal that the DRVSIYGWG and ALLSISSF peptides were able to cut the expression of the proinflammatory cytokine genes iNOS, IL-6, TNF-α, and COX-2 in the context of RAW 264.7 macrophages.

Microalgae-based wastewater treatment for developing economic and environmental sustainability: Current status and future prospects.

Over the last several decades, concerns about climate change and pollution due to human activity has gained widespread attention. Microalgae have been proposed as a suitable biological platform to reduce carbon dioxide, a major greenhouse gas, while also creating commercial sources of high-value compounds such as medicines, cosmetics, food, feed, and biofuel. Industrialization of microalgae culture and valorization is still limited by significant challenges in scaling up the production processes due to economic constraints and productivity capacities. Therefore, a boost in resource usage efficiency is required. This enhancement not only lowers manufacturing costs but also enhancing the long-term viability of microalgae-based products. Using wastewater as a nutrient source is a great way to reduce manufacturing costs. Furthermore, water scarcity is one of the most important global challenges. In recent decades, industrialization, globalization, and population growth have all impacted freshwater resources. Moreover, high amounts of organic and inorganic toxins in the water due to the disposal of waste into rivers can have severe impacts on human and animal health. Microalgae cultures are a sustainable solution to tertiary and quaternary treatments since they have the ability to digest complex contaminants. This review presents biorefineries based on microalgae from all angles, including the potential for environmental pollution remediation as well as applications for bioenergy and value-added biomolecule production. An overview of current information about microalgae-based technology and a discussion of the associated hazards and opportunities for the bioeconomy are highlighted.

Discovery of calcium-binding peptides derived from defatted lemon basil seeds with enhanced calcium uptake in human intestinal epithelial cells, Caco-2.

It is anticipated that calcium-chelating peptides may serve to enhance the absorption of calcium. This research examined defatted lemon basil seeds (DLBS) which had been treated with Alcalase under optimized parameters for the degree of hydrolysis for proteolysis, discovering that the activity for calcium-binding in a competitive condition with phosphate ion was 60.39 ± 1.545%. The purification of the hydrolysates was performed via ultrafiltration along with reversed-phase high performance liquid chromatography (RP-HPLC). Determination of the purified peptide amino acid sequence was confirmed for both peptides and reported as Ala-Phe-Asn-Arg-Ala-Lys-Ser-Lys-Ala-Leu-Asn-Glu-Asn (AFNRAKSKALNEN; Basil-1), and Tyr-Asp-Ser-Ser-Gly-Gly-Pro-Thr-Pro-Trp-Leu-Ser-Pro-Tyr (YDSSGGPTPWLSPY; Basil-2). The respective activities for calcium-binding were 38.62 ± 1.33%, and 42.19 ± 2.27%. Fluorescence spectroscopy, and fourier transform infrared spectroscopy were employed in order to assess the chelating mechanism between calcium and the peptides. It was found that the calcium ions took place through the activity of the amino nitrogen atoms and the oxygen atoms on the carboxyl group. Moreover, both of these peptides served to improve calcium transport and absorption in Caco-2 cell monolayers, depending on the concentration involved. It was revealed that the peptide-calcium complexes offered an increased calcium absorption percentage when compared to free calcium at similar concentrations. It might be concluded that the peptide within the peptide-calcium complex can promote calcium absorption through both active and passive transport pathways by increasing calcium concentration and promoting cell membrane interaction. Accordingly, DLBS protein can be considered a strong potential source of protein which can be used to produce calcium-binding peptides and might therefore play a role in the production of nutraceutical foods as a bioactive ingredient.

A novel angiotensin I-converting enzyme inhibitory peptide derived from the trypsin hydrolysates of salmon bone proteins.

When fish are processed, fish bone becomes a key component of the waste, but to date very few researchers have sought to use fish bone to prepare protein hydrolysates as a means of adding value to the final product. This study, therefore, examines the potential of salmon bone, through an analysis of the benefits of its constituent components, namely fat, moisture, protein, and ash. In particular, the study seeks to optimize the process of enzymatic hydrolysis of salmon bone with trypsin in order to produce angiotensin-I converting enzyme (ACE) inhibitory peptides making use of response surface methodology in combination with central composite design (CCD). Optimum hydrolysis conditions concerning DH (degree of hydrolysis) and ACE-inhibitory activity were initially determined using the response surface model. Having thus determined which of the salmon bone protein hydrolysates (SBPH) offered the greatest level of ACE-inhibitory activity, these SBPH were duly selected to undergo ultrafiltration for further fractionation. It was found that the greatest ACE-inhibitory activity was achieved by the SBPH fraction which had a molecular weight lower than 0.65 kDa. This fraction underwent further purification using RP-HPLC, revealing that the F7 fraction offered the best ACE-inhibitory activity. For ACE inhibition, the ideal peptide in the context of the F7 fraction comprised eight amino acids: Phe-Cys-Leu-Tyr-Glu-Leu-Ala-Arg (FCLYELAR), while analysis of the Lineweaver-Burk plot revealed that the FCLYELAR peptide can serve as an uncompetitive ACE inhibitor. An examination of the molecular docking process showed that the FCLYELAR peptide was primarily able to provide ACE-inhibitory qualities as a consequence of the hydrogen bond interactions taking place between ACE and the peptide. Furthermore, upon isolation form the SBPH, the ACE-inhibitory peptide demonstrated ACE-inhibitory capabilities in vitro, underlining its potential for applications in the food and pharmaceutical sectors.

A modified approach for high-quality RNA extraction of spore-forming Bacillus subtilis at varied physiological stages.

BACKGROUND: High quality RNA is required for the molecular study. Sample preparation of the spore-forming, Gram-positive bacteria like Bacillus sp., remains challenging although several methods have been proposed. Those techniques were simply developed using cell samples at certain growth stages despite some molecular studies like transcriptomic analyses require RNA samples from different physiological stages. METHODS AND RESULTS: We developed the rapid, simple yet effective cell-lysis technique with limit use of harsh reagents by modifying the kit-based protocols. Appropriate lysozyme loading (20 mg/mL), incubation time (30 min), and temperature (37 °C) enabled cell lysis and enhanced RNA extraction from both vegetative cells and endospores of Bacillus subtilis TL7-3. High RNA Integrity Numbers and ratios of A260/A280 and A260/A230 of all RNA products collected during the batch cultivation confirmed that invert mixing with absolute ethanol prevented RNA damage during protein denaturation. With the process modification of the major steps in cell lysis and RNA extraction compared with the kit-based protocols that are typically used in laboratory work, interestingly, our modified protocol, simple-yet-effective, yielded higher concentration, purity, and integrity of RNA products from all cell samples collected at different physiological stages. While the kit-based protocols either failed to provide high RNA concentration or RNA purity and integrity for all cell samples particularly during the late-log, stationary, or sporulation. CONCLUSIONS: Therefore, we can claim the significance of this modified protocol to be applicable for RNA extraction to those spore-forming Gram-positive bacteria not limited to B. subtilis growing at varied physiological stages.

Antioxidant properties of peptides obtained from the split gill mushroom (Schizophyllum commune).

This study sought to assess the ideal conditions under which hydrolysate can be produced from the split gill mushroom proteins through the microbial protease, Alcalase. The research employed a central composite design and response surface methodology. Three specific parameters were varied for the purposes of the experimental process, while a fixed pH value of 8 was used in all cases. The variables were hydrolysis temperature (set as 45 °C, 50 °C, or 55 °C), hydrolysis time (set as 60 min, 120 min, or 180 min), and the ratio of enzyme to substrate (set as 2%, 4%, or 6% w/v). The variables under investigation exert a significant influence upon degree of hydrolysis (DH) in addition to 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity (p < 0.05). Fractionation of the hydrolysate was accomplished using molecular weight (MW) cut-off membranes, while the greatest radical-scavenging capability was observed in the < 0.65 kDa fraction. The MW < 0.65 kDa fraction underwent separation through RP-HPLC in order to create five sub-fractions. Among these, the greatest ABTS radical-scavenging capability was observed in the F5 sub-fraction, which was therefore chosen to undergo additional examination using quadrupole-time-of-flight-electron spin induction-mass spectrometry-based de novo peptide sequencing. Via this process it was possible to determine five antioxidant peptides. Furthermore, the MW < 0.65 kDa fraction was able to demonstrating cellular antioxidant activity in the context of a human intestinal cancer cell line (HT-29). The extent of this activity was shown to depend upon the concentration levels of the peptide.

Hydrolysates from bee pollen could induced apoptosis in human bronchogenic carcinoma cells (ChaGo-K-1).

In order to examine bee pollen hydrolysates to assess their anticancer and antioxidant properties, hydrolysis of bee pollen was first performed using three different commercially available enzymes: Alcalase®, Neutrase®, and Flavourzyme®. The study used DPPH and ABTS assay to evaluate the antioxidant properties of the hydrolysates obtained from bee pollen. All of the tested hydrolysates demonstrated antioxidant activity, while hydrolysate based on Alcalase® offered a high value for IC50 and was therefore chosen for further separation into five sub-fractions via ultrafiltration. The greatest antioxidant activity was presented by the MW < 0.65 kDa fraction, which achieved an IC50 value of 0.39 ± 0.01 µg/mL in the DPPH assay and 1.52 ± 0.01 µg/mL for ABTS. Purification of the MW < 0.65 kDa fraction was completed using RP-HPLC, whereupon the three fractions from the original six which had the highest antioxidant activity underwent further examination through ESI-Q-TOF-MS/MS. These particular peptides had between 7 and 11 amino acid residues. In the case of the MW < 0.65 kDa fraction, testing was also carried out to determine the viability of lung cancer cell lines, represented by ChaGo-K1 cells. Analysis of the antiproliferative properties allowed in vitro assessment of the ChaGo-K1 cells' viability following treatment using the MW < 0.65 kDa fraction. Flow-cytometry generated date which revealed that it was possible for the MW < 0.65 kDa fraction to induce apoptosis in the ChaGo-K1 cells in comparison to the results with cells which had not been treated.

Angiotensin-I converting enzyme inhibitory peptide derived from the shiitake mushroom (Lentinula edodes).

ABSTRACT: Angiotensin-I converting enzyme (ACE) inhibitors are widely used to control hypertension. In this study, protein hydrolysates from shiitake mushroom were hydrolyzed to prepare ACE-inhibitory peptides. Optimum process conditions for the hydrolysis of shiitake mushrooms using Alcalase were optimized using response surface methodology. Monitoring was conducted to check the degree of hydrolysis (DH) and ACE inhibitory activity. In the results, the optimum condition with the highest DH value of 28.88% was 50.2 °C, 3-h hydrolysis time, and 1.16 enzyme/substrate ratios. The highest ACE inhibitory activity (IC50 of 0.33 µg/mL) was under 47 °C, 3 h 28 min hydrolysis time, and 0.59 enzyme/substrate ratios. The highest activity was fractionated into 5 ranges of molecular weight, and the fraction below 0.65 kDa showed the highest activity with IC50 of 0.23 µg/mL. This fraction underwent purification using RP-HPLC, meanwhile the peak which offered a retention time of about 37 min showed high ACE inhibitory activity. Mass spectrometry identified the amino acid sequence of this peak as Lys-Ile-Gly-Ser-Arg-Ser-Arg-Phe-Asp-Val-Thr (KIGSRSRFDVT), with a molecular weight of 1265.43 Da. The synthesized variant of this peptide produced an ACE inhibitory activity (IC50) of 37.14 µM. The peptide KIGSRSRFDVT was shown to serve as a non-competitive inhibitor according to the Lineweaver-Burk plot findings. A molecular docking study was performed, which showed that the peptide binding occurred at an ACE non-active site. The findings suggest that peptides derived from shiitake mushrooms could serve either as useful components in pharmaceutical products, or in functional foods for the purpose of treating hypertension.

An in vitro study of lipase inhibitory peptides obtained from de-oiled rice bran.

De-oiled rice bran (DORB) is a potentially useful by-product of the rice bran oil industry. DORB may prove to be an important protein source, and also contains many other micronutrients. This study has the principal aim of optimizing the process of DORB protein hydrolysate preparation, and then testing the hydrolysate to determine its lipase inhibitory activity. DORB underwent hydrolysis using Alcalase® and response surface methodology (RSM). The resulting degree of hydrolysis (DH) was then monitored along with the extent of any lipase inhibitory activity. The optimum levels of lipase inhibition were obtained at a temperature of 49.88 °C, a duration of 150.43 minutes, and 1.53% Alcalase® used for the sample 5% (w/v) solution. In these conditions, the DH value was 35.65%, and the IC50 value for lipase inhibitory activity was 2.84 µg mL-1. Five ranges of different molecular weights were obtained via fractionation, whereupon it was determined that the highest level of inhibitory activity was achieved by the <0.65 kDa fraction. This fraction was then further purified via RP-HPLC, and the resulting peak had a retention time of 21.75 minutes (F 2 sub-fraction) and exhibited high lipase inhibitory activity. Mass spectrometry was used to determine the amino acid sequence for this peak, identified as FYLGYCDY. This particular peptide is categorized as bitter, with a non-toxic profile, and having poor water solubility. The synthesized form of this peptide showed lipase inhibitory activity measured by an IC50 value of 0.47 ± 0.02 µM. The Lineweaver-Burk plot revealed that FYLGYCDY is a non-competitive inhibitor, while analysis of the docking results provided details of the FYLGYCDY peptide binding site with the porcine pancreatic lipase (PPL) complex, which is a competitive type. It can be inferred from these findings that DORB may prove a useful raw material source for the production of anti-obesity peptides which might enhance the therapeutic and commercial performance of functional foods and healthcare products.

ACE inhibitory peptides derived from de-fatted lemon basil seeds: optimization, purification, identification, structure-activity relationship and molecular docking analysis.

The oil processing industry generates significant quantities of lemon basil seed residue which is not currently used to any significant extent. However, this by-product has important potential as a source of bioactive peptides which may play a role as ingredients in functional foods. This study therefore sought to optimize the preparation techniques used to obtain the necessary protein hydrolysate from de-fatted lemon basil seeds (DLBS), and subsequently to examine the ACE inhibitory capabilities of the resulting hydrolysate. Response Surface Methodology (RSM) was used for the hydrolysis of DLBS by Alcalase®, with observation of the resulting ACE inhibitory activity and degree of hydrolysis (DH). The optimum conditions were 55 °C and 103 minutes with a ratio of enzyme to substrate of 7.0% w/v. The hydrolysate was fractionated by ultrafiltration and purified through RP-HPLC. The results reveal that the F2 sub-fraction demonstrated the highest ACE inhibitory activity. The amino acid sequence of this peak was identified by mass spectrometry as LGRNLPPI and GPAGPAGL with a molecular weight of 879.06 and 639.347 Dalton, respectively. These peptides were classified as non-toxic and bitter peptides. For the synthesized version of these peptides, the ACE inhibitory activity values, measured by IC50, were 0.124 ± 0.02 mM and 0.013 ± 0.001 mM, respectively. Analysis of the Lineweaver-Burk plot confirmed that these peptides served as non-competitive ones. The study of molecular docking showed that the ACE inhibitory behavior of both purified peptides was mainly due to the interactions of the hydrogen bonds between the peptides and ACE. It is therefore suggested that DLBS may be a useful raw material allowing the production of antihypertensive peptides which can offer therapeutic and commercial benefits as an ingredient in functional foods.

Peptides obtained from edible mushrooms: Hericium erinaceus offers the ability to scavenge free radicals and induce apoptosis in lung cancer cells in humans.

This research examined the antioxidant abilities of peptides derived from the Hericium erinaceus mushroom produced via three microbial proteases (Alcalase®, Neutrase®, and Flavourzyme®) at varying concentrations of 10, 25, and 50 mg mL-1. The H. erinaceus mushroom hydrolysate produced from 10 mg mL-1 Alcalase® with 4 hours' digestion resulted in the highest levels of 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazl (DPPH), and nitric oxide (NO) radical scavenging activities. Fractionation of this particular hydrolysate was then performed by ultrafiltration (10, 5, 3, and 0.65 kDa molecular weight cut-off membranes). The MW < 0.65 kDa fraction demonstrated the highest level of radical scavenging activity in the context of ABTS, DPPH, and NO. Therefore, the fraction MW < 0.65 kDa was performed with RP-HPLC to obtain the four principal fractions termed F1-4. The results reveal that the F4 sub-fraction demonstrated the best radical scavenging ability. Additionally, the F4 sub-fraction was able to reduce the potential for DNA damage caused by hydroxyl radicals. This was revealed by testing using the Fenton reaction and the pUC19, pKS, and pBR322 plasmids. This outcome was demonstrated through in vitro antiproliferative activity in human lung carcinoma cell lines (Chago-K1) using MTT assay. The F4 sub-fraction was also demonstrated to induce apoptosis, as indicated using the FITC Annexin V apoptosis detection kit with PI. Furthermore, it was also found that the activity of caspase-3, -8, and -9 in Chago-K1 cells was enhanced after exposure periods of 24 and 48 hours. Finally, this sub-fraction was selected for peptide sequencing via quadrupole-time-of-flight-electron spin induction-mass spectrometry-based de novo peptide sequencing.

The apoptotic and free radical-scavenging abilities of the protein hydrolysate obtained from chicken feather meal.

This study examined the antioxidant capabilities of peptides derived from chicken feather meal (CFM) protein hydrolysates which were produced using 3 different microbial proteases (Neutrase, Alcalase, and flavourzyme) and tested at varying concentrations, namely 1, 2, and 5% by weight. The highest levels of 2,2-diphenyl-1-picrylhydrazl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging activities were presented by CFM hydrolysate derived using 5 wt% Neutrase and digested for 4 h. Fractionation of this particular hydrolysate was then performed by applying 10, 5, 3, and 0.65 kDa molecular weight cutoff membranes. It was then determined that the molecular weight (MW) < 0.65 kDa fraction achieved the greatest level of free radical scavenging activity in the context of DPPH and ABTS. The MW < 0.65 kDa fraction then underwent additional fractionation using reverse-phase high-performance liquid chromatography to derive 3 main fractions designated as F1, F2, and F3. All of these fractions presented a high level of activity in DPPH radical scavenging, although no significant ABTS scavenging was observed. Quadrupole time-of-flight tandem mass spectrometry was used in determining the peptide contents of the fractions as Phe-Asp-Asp-Arg-Gly-Arg-X for F1 (FDDRGRX, 875 Da), Val-Thr-Leu-Ala-Val-Thr-Lys-His for F2 (VTLAVTKH, 868 Da), and Val-Ser-Glu-Ile-X-Ser-Ile-Pro-Ile-Ser for F3 (VSEIXSIPIS, 1,055 Da). Moreover, the F2 fraction was shown to be capable of preventing DNA damage induced by hydroxyl radicals, as indicated in tests using the plasmids pKS, pUC19, and pBR322 via the Fenton reaction. This outcome was demonstrated through in vitro antiproliferative activity in human cell lines based on SW620 colon cancer, using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The F2 fraction at 0.5 wt.% was also shown to be capable of inducing weak early apoptosis, which could be measured by using the Fluorescein isothiocyanate Annexin V Apoptosis Detection Kit with Propidium Iodide Solution. Furthermore, an increase in caspase-3 and caspase-8 activity was observed in SW620 cells following exposure for 24 h and 48 h.

Two novel ACE inhibitory peptides isolated from longan seeds: purification, inhibitory kinetics and mechanisms.

Angiotensin converting enzyme (ACE) inhibition offers a useful means of managing hypertension, because ACE inhibitors (ACEIs) are known to serve as agents with antihypertensive properties in addition to generating positive metabolic and cardioprotective outcomes. However, current ACEIs are linked to adverse consequences, and so there is a requirement for effective but safer compounds, which might be achieved through chemical synthesis or the isolation of naturally obtained bioactive molecules. Protein hydrolysates with ACEI activity can be produced by the combined pepsin and pancreatin proteolysis (to mimic gastrointestinal digestion) of longan seed protein. This study examined longan seed protein hydrolysates, obtained from a sequential 3 h digestion with pepsin and then pancreatin. The resulting hydrolysate underwent sequential ultrafiltration membrane fractionation with a 10, 5, and 3 kDa molecular weight cut-off (MWCO). The permeate derived from the <3 kDa MWCO demonstrated the highest ACEI activity. This permeate subsequently underwent separation by reverse-phase high performance liquid chromatography to give the main fractions on the basis of differing elution times. The ACEI IC50 values for these fractions were then identified. Quadrupole time-of-flight tandem mass spectrometry was employed to determine the peptide mass for the major peak (F 5), which was shown to be Glu-Thr-Ser-Gly-Met-Lys-Pro-Thr-Glu-Leu (ETSGMKPTEL) and Ile-Ser-Ser-Met-Gly-Ile-Leu-Val-Cys-Leu (ISSMGILVCL). These two peptides were stable over a temperature and pH range of -20 to 90 °C and 2-12, respectively, for 60 min. From the Lineweaver-Burk plot, both peptides inhibited ACE non-competitively. Molecular docking simulation of the peptides with ACE supported the formation of hydrogen bonds by the peptides with the ACE active pockets. This research indicates that it may be possible to use both of these peptides or longan seed protein hydrolysates in order to create ingredients for functional foods, or to produce pharmaceutical products, capable of lowering hypertension.

The antioxidant potential of peptides obtained from the spotted babylon snail (Babylonia areolata) in treating human colon adenocarcinoma (Caco-2) cells.

This research study investigated the free radical-scavenging activities of peptides which were obtained from the protein hydrolysates of the spotted babylon snail using a combination of pepsin and pancreatin proteolysis which can replicate the conditions of gastrointestinal digestion. In this study, spotted babylon protein hydrolysate (SPH) derived from a sequential 3 hour digestion, first with pepsin and then with pancreatin, was examined. SPH was fractionated using molecular weight cut-off membranes for 10 kDa, 5 kDa, 3 kDa, and 0.65 kDa. It was found that the MW < 0.65 kDa fraction provided the greatest levels of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazl (DPPH), and nitric oxide (NO) radical scavenging activity. Three subfractions of the MW < 0.65 kDa fraction were then generated via RP-HPLC. The subfraction which subsequently demonstrated the greatest free radical scavenging activity was F3, which was accordingly chosen for further investigation commencing with quadrupole-time-of-flight-electron spin induction-mass spectrometry-based de novo peptide sequencing. This resulted in the identification of a pair of novel peptides: His-Thr-Tyr-His-Glu-Val-Thr-Lys-His (HTYHEVTKH), and Trp-Pro-Val-Leu-Ala-Tyr-His-Phe-Thr (WPVLAYHF). The WPVLAYHF peptide exhibited greater antioxidant activity. The study also confirmed that the F3 sub-fraction was able to prevent hydroxyl radicals from causing DNA damage by conducting tests which involved the pKS, pUC19, and pBR322 plasmids using the Fenton reaction. In addition, cellular antioxidant activity was demonstrated by two synthetic peptides toward the human adenocarcinoma colon (Caco-2) cell line, with the potency of the activity dependent upon the peptide concentration.