Crude Hydrolysate Research Articles

Identification of New Angiotensin-Converting Enzyme Inhibitory Peptides Isolated from the Hydrolysate of the Venom of Nemopilema nomurai Jellyfish.

Recently, jellyfish venom has gained attention as a promising reservoir of pharmacologically active compounds, with potential applications in new drug development. In this investigation, novel peptides, isolated from the hydrolysates of Nemopilema nomurai jellyfish venom (NnV), demonstrate potent inhibitory activities against angiotensin-converting enzyme (ACE). Proteolytic enzymes-specifically, papain and protamex-were utilized for the hydrolysis under optimized enzymatic conditions, determined by assessing the degree of hydrolysis through the ninhydrin test. Comparative analyses revealed that papain treatment exhibited a notably higher degree of NnV hydrolysis compared to protamex treatment. ACE inhibitory activity was quantified using ACE kit-WST, indicating a substantial inhibitory effect of 76.31% for the papain-digested NnV crude hydrolysate, which was validated by captopril as a positive control. The separation of the NnV-hydrolysate using DEAE sepharose weak-anion-exchange chromatography revealed nine peaks under a 0-1 M NaCl stepwise gradient, with peak no. 3 displaying the highest ACE inhibition of 96%. The further purification of peak no. 3 through ODS-C18 column reverse-phase high-performance liquid chromatography resulted in five sub-peaks (3.1, 3.2, 3.3, 3.4, and 3.5), among which 3.2 exhibited the most significant inhibitory activity of 95.74%. The subsequent analysis of the active peak (3.2) using MALDI-TOF/MS identified two peptides with distinct molecular weights of 896.48 and 1227.651. The peptide sequence determined by MS/MS analysis revealed them as IVGRPLANG and IGDEPRHQYL. The docking studies of the two ACE-inhibitory peptides for ACE molecule demonstrated a binding affinity of -51.4 ± 2.5 and -62.3 ± 3.3 using the HADDOCK scoring function.

Engineering microbial division of labor for plastic upcycling

Plastic pollution is rapidly increasing worldwide, causing adverse impacts on the environment, wildlife and human health. One tempting solution to this crisis is upcycling plastics into products with engineered microorganisms; however, this remains challenging due to complexity in conversion. Here we present a synthetic microbial consortium that efficiently degrades polyethylene terephthalate hydrolysate and subsequently produces desired chemicals through division of labor. The consortium involves two Pseudomonas putida strains, specializing in terephthalic acid and ethylene glycol utilization respectively, to achieve complete substrate assimilation. Compared with its monoculture counterpart, the consortium exhibits reduced catabolic crosstalk and faster deconstruction, particularly when substrate concentrations are high or crude hydrolysate is used. It also outperforms monoculture when polyhydroxyalkanoates serves as a target product and confers flexible tuning through population modulation for cis-cis muconate synthesis. This work demonstrates engineered consortia as a promising, effective platform that may facilitate polymer upcycling and environmental sustainability.

Screening of yeast co‐culture using crude hydrolysate for co‐fermentation of pentose and hexose

AbstractThe use of all sugars from biomass fractionation – that is, hexoses and pentoses – is essential to develop feasible processes in a biorefinery context. Hexoses are converted into different products by most microorganisms. The main issue is the conversion of pentoses, especially when mixed with hexoses. Improving sugar conversion in co‐fermentation is a major challenge, which was explored in this work. Co‐culture using Saccharomyces cerevisiae and evolved Kluyveromyces marxianus strains was evaluated by screening using 24 deep‐well plates. Glucose depletion and 83% of xylose conversion were achieved with supplemented hemicellulosic hydrolysate medium (medium volume corresponding to 2/5 of deep‐well volume), and initial cell concentrations of 2.5 g L‐1 for each yeast, incubated at 150 rpm. The screening strategy provided reliable data that confirmed the co‐culture as a promising approach for the co‐assimilation of biomass‐derived sugars. The importance of aeration control for effective xylose assimilation was also pointed out.

Microwave-assisted cassava pulp hydrolysis as food waste biorefinery for biodegradable polyhydroxybutyrate production.

Cassava pulp is one of the most abundant agricultural residues that can cause serious disposal problems. This study aimed to apply a biorefinery approach by examining the feasibility of microwave-assisted cassava pulp hydrolysis to attain sustainable management and efficient use of natural resources. Four factors, namely, the liquid-to-solid ratio (20mL/g, 10mL/g, 7.5mL/g, and 5mL/g), types of acids (H2SO4 and H3PO4), watt power (600W, 700W, and 800W) and time (3, 5 and 8min), were carefully investigated. The highest fermentable sugar content of 88.1g/L ± 0.7g/L (0.88g fermentable sugars/g dry cassava pulp) was achieved when 20mL/g cassava pulp was hydrolyzed with 2.5% (v/v) H2SO4 under microwave irradiation at 800W for 8min. Glucose was a major product (82.0g/L ± 5.2g/L). The inhibitor concentration was 5.17g/L ± 0.01g/L, and the levulinic acid concentration was 5.15g/L ± 0.01g/L. The results indicated that the liquid-to-solid ratio, diluted acid concentration, irradiation watt power and time were important factors in producing fermentable sugars from acid hydrolysis under microwave irradiation. The crude hydrolysate was used for PHB production by Cupriavidus necator strain A-04. The hydrolysate to nutrients ratio of 30:70 (v/v) yielded a cell dry weight of 7.5g/L ± 0.1g/L containing PHB content of 66.8% ± 0.3% (w/w), resulting in a yield (g-PHB/g- ) of 0.35g/g. This study demonstrated that the microwave-assisted cassava pulp hydrolysate developed in this study provided a high amount of glucose (88.1% conversion) and resulted in a low concentration of inhibitors without xylose; this was successfully achieved without pregelatinization, alkaline pretreatment or detoxification.

Enhancing the bioconversion of ferulic acid from alkaline hydrolysate of corn cobs to vanillin by Amycolatopsis thermoflava under nutrient limitation and reducing sugar control

AbstractBACKGROUNDThe use of commercially available ferulic acid (FA) as a precursor for vanillin production is costly. The utilization of agro‐waste as a starting material provides an alternative to obtain FA at a low cost and establish an economically profitable production system. In this work, optimization of ultrasound‐assisted extraction conditions for releasing FA from corn cobs using alkaline treatment was conducted. Further, bioconversion of FA from the crude hydrolysate of corn cobs to vanillin using Amycolatopsis thermoflava was performed.RESULTSSince autoclaved hydrolysate was unfavorable for cell growth and vanillin formation, pasteurization was proposed. However, the presence of contaminants limited the entire process. Aiming to suppress the growth of contaminants and increase vanillin yield, novel strategies of limiting and controlling the nutrients for the strain were developed. Optimal hydrolysis conditions (NaOH concentration = 0.5 M, solid loading = 10% for 30 min) produced hydrolysate with relatively high titers of FA (715 mg L−1) and p‐coumaric acid (1025 mg L−1). To improve vanillin production from FA, four parameters (including the effects of sterilization method, nutrient limitation, initial biomass concentration, and reducing sugar control) were investigated. By adopting these strategies and performing bioconversion of FA to vanillin with an initial biomass concentration of 1.5 g L−1, the yield of vanillin was significantly increased by 29.5‐fold to 477.4 mg g−1, compared to bioconversion using autoclaved hydrolysate.CONCLUSIONThis approach represents a relatively economical method for vanillin production from agro‐waste because it uses crude hydrolysate, requires minimum nutrients, and involves non‐modified organisms with a relatively short bioconversion time (17 h). © 2022 Society of Chemical Industry (SCI).

Producing aromatic amino acid from corn husk by using polyols as intermediates

Agricultural biomass remains as one of the commonly found waste on Earth. Although valorisation of these wastes has been studied in detail, the fermentation-based processes still need improvement due to the high cost of hydrolysing enzymes, and the presence of growth inhibitors which constrains the fermentation to produce high-value products. To address these challenges, we developed an integrated process in this study combining abiotic- and bio-catalysis to produce l-tyrosine from corn husk. The first step involved a one-pot hydrolytic hydrogenation tandem reaction without the use of the expensive enzymes, which yielded a mixture of polyols and sugars. Without any purification, these crude hydrolysates can be almost completely utilized by an engineered Escherichia coli strain, which did not exhibit any growth inhibition. The strain produced 0.44 g/L l-tyrosine from 10 g/L crude corn husk hydrolysates, demonstrating the feasibility of converting agricultural biomass into a valuable aromatic amino acid via an integrated process.

Cell Immobilization Using Alginate-Based Beads as a Protective Technique against Stressful Conditions of Hydrolysates for 2G Ethanol Production.

The development of biorefineries brings the necessity of an efficient consumption of all sugars released from biomasses, including xylose. In addition, the presence of inhibitors in biomass hydrolysates is one of the main challenges in bioprocess feasibility. In this study, the application of Ca-alginate hybrid gels in the immobilization of xylose-consuming recombinant yeast was explored with the aim of improving the tolerance of inhibitors. The recombinant yeast Saccharomyces cerevisiae GSE16-T18SI.1 (T18) was immobilized in Ca-alginate and Ca-alginate–chitosan hybrid beads, and its performance on xylose fermentation was evaluated in terms of tolerance to different acetic acid concentrations (0–12 g/L) and repeated batches of crude sugarcane bagasse hemicellulose hydrolysate. The use of the hybrid gel improved yeast performance in the presence of 12 g/L of acetic acid, achieving 1.13 g/L/h of productivity and reaching 75% of the theoretical ethanol yield, with an improvement of 32% in the xylose consumption rate (1:1 Vbeads/Vmedium, 35 °C, 150 rpm and pH 5.2). The use of hybrid alginate–chitosan gel also led to better yeast performance at crude hydrolysate, yielding one more batch than the pure-alginate beads. These results demonstrate the potential of a hybrid gel as an approach that could increase 2G ethanol productivity and allow cell recycling for a longer period.

A multi-omics approach to assess production of the valuable peptides and amino acids in porcine blood protein hydrolysate

Animal blood is an unutilized byproduct of the meat processing industry that could potentially be exploited as a bioresource. This study analyzed the enzymatic hydrolysates of porcine whole blood for the possible production of amino acids. Porcine whole blood was hydrolyzed by endo- and exo-protease cocktails, and the crude hydrolysate filtered and a dried powder generated. The peptide contents, free amino acids, and their derivative compositions, diaminopeptides, residual antibiotics, and microbiome communities were then analyzed. The proteolysis reaction converted proteins with a molecular mass <750 m/z to peptides and enhanced free-form amino acids. LC-MS-based metabolomics showed that leucine and glutamic acid contents were higher in the blood hydrolysate than other amino acids. Furthermore, in the proteolyzed whole blood there were diverse bacterial species, predominantly from the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Blood byproducts could thus potentially be utilized to provide valuable compounds for bioactive food supplements.

An exploration of angiotensin-converting enzyme (ACE) inhibitory peptides derived from gastrointestinal protease hydrolysate of milk using a modified bioassay-guided fractionation approach coupled with in silico analysis

An improved bioassay-guided fractionation was performed to effectively screen angiotensin-I converting enzyme inhibitory (ACEI) peptides from milk protein hydrolysate. The aqueous normal phase liquid chromatography, namely hydrophilic interaction liquid chromatography (HILIC), was used as a format of solid-phase extraction (SPE) short column for the first fractionation, then the HILIC-SPE fraction with the best ACEI activity (IC50 = 61.75 ± 5.74 µg/mL; IC50 = half-maximal inhibitory concentration) was obtained when eluted by 95% acetonitrile + 0.1% formic acid (fraction F1). The best HILIC-SPE fraction was further fractionated using reversed-phase (RP)-SPE short column. The best RP-SPE fraction was obtained when eluted by 20% acetonitrile + 0.1% formic acid (fraction P3) with an ACEI activity of IC50 36.22 ± 1.18 µg/mL. After the 2-step fractionation, the IC50 value of fraction P3 significantly decreased by 8.92-fold when compared with the crude hydrolysate. Several peptides were identified from fraction P3 using liquid chromatography-tandem mass spectrometry. The in silico analysis of these identified sequences based on the BIOPEP database predicted that HLPLPLL (HL-7) was the most active peptide against angiotensin-converting enzyme (ACE). The HL-7 derived from β-casein showed a potent ACEI activity (IC50 value is 16.87 ± 0.3 µM). The contents of HL-7 in the gastrointestinal protease hydrolysate and RP-SPE fraction originated from 1 mg of milk proteins were quantified using a multiple reaction monitoring mode upon liquid chromatography-tandem mass spectrometry analysis to give 19.86 ± 1.14 pg and 14,545.8 ± 572.9 pg, respectively. Besides, the kinetic study indicated that HL-7 was a competitive inhibitor and the result was rationalized using the docking simulation. The study demonstrated an efficient screening of ACEI peptides from commercially available milk powders using a simple SPE process instead of a sophisticated instrument such as HPLC. Moreover, the potent ACEI peptide HL-7 uncovered by this method could be a natural ACE inhibitor.

Integrated biorefinery development using winery waste streams for the production of bacterial cellulose, succinic acid and value-added fractions

An integrated biorefinery has been developed using winery wastes (grape pomace-GP, stalks-GS, wine lees-WL). Bacterial cellulose was produced from GP extracted free sugars. Grape-seed oil and polyphenols were extracted from GP. Experimental design was employed to optimize lignin removal (50.8%) from mixtures of remaining GP solids and GS via NaOH (1.19% w/v) treatment at 70°C for 30 min. Delignification liquid contained condensed tannins with 76% Stiasny number. Enzymatic hydrolysis produced a sugar-rich hydrolysate (40.2 g/L sugars). Ethanol, antioxidants, tartaric acid and nutrient-rich hydrolysate were produced from WL. The crude hydrolysates were used in fed-batch Actinobacillus succinogenes cultures for 37.2 g/L succinic acid production. The biorefinery produces 42.65 g bacterial cellulose, 24.3 g oil, 40.3 g phenolic-rich extract with 1.41 Antioxidant Activity Index, 80.2 g ethanol, 624.8 g crude tannin extract, 20.03 g tartaric acid and 157.8 g succinic acid from 1 kg of each waste stream.

Chemical-free recovery of crude protein from livestock manure digestate solid by thermal hydrolysis

Protein is becoming an increasingly important resource for a variety of commercial applications. Yet, a large volume of protein is being wasted. Notably, livestock manure solids have a significant content of protein which is not only underutilized, but prone to runoff and eventual breakdown to reactive nitrogen compounds, contributing to eutrophication. It would be desirable to remove protein before it causes environmental hazards and then convert it to value-added commercial applications. We have developed a novel thermal hydrolysis process (THP) to extract crude protein from livestock manure solid, or manure digestate solid (MDS) in particular, without the use of any chemical. We demonstrate the versatility of our new process to control the molecular weight (MW) distribution of the extracted protein hydrolysate (PH). The antioxidant activity of the crude protein hydrolysate (CPH) has been examined through Oxygen Radical Absorbance Capacity Assay. The results have shown that our CPH had its antioxidant capacity against the peroxyl radical similar to that of vitamin E and exhibited almost 7 times as strong inhibition against the hydroxyl radical as vitamin E. We also evaluated the nutritional value of our PH by analyzing its amino acid composition and the MW distribution through amino acid analysis, SDS-PAGE, and MALDI-TOF mass spectroscopy. The characterizations have revealed that the PH recovered from MDS had 2.5 times as much essential amino acids as soybean meal on dry matter basis, with the MW distribution ranging from over a 100 Da to 100 KDa. Finally, the protein powder was prepared from the extracted CPH solution and its composition was analyzed.

Bioprocess development using organic biowaste and sustainability assessment of succinic acid production with engineered Yarrowia lipolytica strain

The utilization of crude waste streams and the development of novel bioprocessing routes are necessary in order to expand the industrial implementation of bio-based succinic acid production. This work focuses on the utilization of zero cost municipal organic biowaste for the production of bio-based succinic acid using the engineered yeast strain Yarrowia lipolytica PSA02004. Fermentation advances focused on pH regulation towards reduced NaOH requirements. Optimum initial carbon source concentration (100–120 g/L) in crude hydrolysates was estimated in shake flask cultures. Optimum kLα (183.2 h−1) in fed-batch cultures resulted in 42.2 g/L succinic acid with 0.38 g/g yield and 0.84 g/L/h productivity. A two-stage pH regulation strategy was employed to enhance succinic acid production efficiency and reduce NaOH requirements. The gradual reduction of pH from 6 to 5.5 resulted in 54.4 g/L succinic acid with 0.44 g/g yield and 0.82 g/L/h productivity and 43% lower NaOH consumption. The minimum selling price of succinic acid was estimated at $2.7/kg and $2.94/kg at annual production capacity of 50,000 t when the production cost of organic biowaste derived sugars were considered at $10/tTS and $110/tTS, respectively. The global warming potential and abiotic depletion potential were estimated at 2.72 kgCO2-eq/kgSA and 33.4 MJ/kgSA, respectively. Results showed that pH regulation is an important operating parameter towards the sustainable succinic acid production using municipal organic biowaste.

Valorisation of grape stalks and pomace for the production of bio-based succinic acid by Actinobacillus succinogenes

Valorisation of food supply chain waste for the production of bio-based platform chemicals will facilitate the transition to the sustainable bio-economy era. To this end, a novel process has been developed for the conversion of grape pomace and stalks into bio-based succinic acid. Free sugars (2.55 g/100 g), a phenolic-rich extract (185.75 mg gallic acid equivalent per g extract with Antioxidant Activity Index of 1.03) and lipids (8.2 g/ 100 g) were initially extracted from grape pomace. Residual grape pomace solids were subsequently mixed with grape stalks (1:1 ratio) and treated with sodium hydroxide (1%, w/v) for lignin removal. The residual solids were treated with varying H2SO4 concentrations (3–10 %, v/v) followed by enzymatic hydrolysis using different dosages of cellulases and β-glucosidase leading to the release of 22.7 g glucose per 100 g remaining solids (48 % cellulose hydrolysis). Actinobacillus succinogenes fed-batch cultures carried out with the crude hydrolysate as fermentation medium and free sugars from grape pomace as feeding solution resulted in 40.2 g/L succinic acid concentration with a yield of 0.67 g/g and productivity of 0.79 g/L/h. This study proposes for the first time a sustainable process for valorising grape pomace and stalks for efficient bio-based succinic acid production integrated with the extraction of value-added fractions.

Skin-protective properties of peptide extracts produced from white sorghum grain kafirins

In recent years, the cosmetic industry has developed products that provide beneficial functions on the skin, with an increase in the production of cosmeceuticals which has generated great scientific and industrial interest in the search for alternative bioactive ingredients. The objective of this research was to determine the protective effects (antioxidant, anti-inflammatory, and anti-aging) of peptide extracts of the grain of white sorghum [Sorghum bicolor (L.) Moench] against the damage induced by exposure to ultraviolet B irradiation (UVB) in organotypic cultures of human skin. The kafirins (α, β, and γ-kafirin) fraction extracted from the sorghum grain was hydrolyzed with alcalase to produce crude hydrolysates. Then, by ultrafiltration, two peptide extracts with molecular weight 1–3 kDa (PE-3) and < 1 kDa (PE-1) were prepared. Bioassays were performed on organotypic skin cultures (exposed and not exposed to UVB). The results revealed that the treatments with both extracts (PE-3 and PE-1) significantly reduce the damage caused by UVB, by attenuating the depletion of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as by maintaining or increasing the activity of catalase (CAT). Also, PE-3 and PE-1 decreased the levels of pro-inflammatory cytokines such as interleukin 1-β (1L-β), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). Furthermore, PE-3 and PE-1 inhibited collagenase, elastase, and tyrosinase activities. In general, PE-1 exhibited a greater protective effect than PE-3 and like that obtained with glutathione. Therefore, the enzymatic production of PE-3 and PE-1 with protective functions of the skin could be a promising strategy to generate potential ingredients that can be incorporated in new cosmeceutical formulations.

Enzymatic Protein Hydrolysates, and Ultrafiltered Peptide Fractions from Two Molluscs: Tympanotonus fuscatus var. radula (L.) and Pachymelania aurita (M.), with Angiotensin-I-Converting Enzyme Inhibitory and DPPH Radical Scavenging Activities.

Context:Multifunctional food protein-derived peptides attract a great deal of research interest due to their health-promoting benefits. Particularly, peptides that have both antihypertensive and antioxidant properties are desired, since both effects can be synergistic in prevention of cardiovascular diseases.Aim:The aim of this study was to investigate the angiotensin-I-converting enzyme (ACE) inhibitory and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of two species of the Nigerian periwinkles: Pachymelania aurita and Tympanotonus fuscatus.Methods:The ACE inhibitory and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of simulated gastrointestinal digestion (SGID) hydrolysates and ultrafiltered (UF) fractions of T. fuscatus var. radula and P. aurita were determined. Human SGID of the protein extracts of T. fuscatus and P. aurita was carried out using pepsin, trypsin, and chymotrypsin, and the hydrolysates were fractionated into two by centrifugal ultrafiltration. The ACE inhibitory and DPPH radical scavenging activities of the crude hydrolysates and UF fractions were tested. The UF permeates were observed to have relatively higher activities and was subjected to gel filtration chromatography on Sephadex G-50. The chromatographic fractions showed absorbance at 215, 225, and 280 nm and were assayed for DPPH radical scavenging activity.Results:The inhibitory effect of the fractions on ACE activity was reported as the minimum concentration of extract that caused 50% of the inhibition (IC50), where the IC50 values of P. aurita UF permeate and P. aurita UF retentate were 65.2 ± 6.4 and 301.9 ± 59.1 μg/ml, respectively, and that of T. fuscatus UF permeate (TFUFP) and T. fuscatus UF retentate were 54.93 ± 2.83 and 291.7 ± 8.6 μg/ml, respectively.Conclusion:This study suggests the potential health benefits of consuming T. fuscatus var. radula and P. aurita in health maintenance.

In vitro study on novel bioactive peptides with antioxidant and antihypertensive properties from edible rhizomes

Rhizome proteins of white turmeric, turmeric and ginger were hydrolyzed using pepsin and trypsin. The peptides from crude hydrolysates were purified based on antihypertensive and antioxidant activities. The peptide sequences were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The binding energy with ACE of each antihypertensive peptides was predicted by in silico docking. Among the 11 bioactive peptides selected from the three rhizomes, five peptides from ginger harbored both antioxidant and anti-ACE activities. Particularly potent was the VTYM peptide from ginger, with the lowest IC50 and EC50 values of 16.4 ± 0.4 μmol/L, 19.9 ± 2.1 μmol/L and 24.0 ± 3.7 μmol/L in antihypertensive, DPPH, and ABTS assays, respectively. In addition, VTYM was capable of protecting Agrobacterium tumefaciens sodBI sodBII sodBIII triple mutant against menadione treatment. Among turmeric-derived peptides, CGVGAA, DVDP, and CACGGV exhibited strong anti-hypertensive activities, with IC50 values of 18.3 ± 0.1 μmol/L, 19.0 ± 0.6 μmol/L and 25.0 ± 0.2 μmol/L, respectively. Hence, this study demonstrates that many edible rhizomes, apart from serving as valuable sources of essential oil and bioactive metabolites, are also bountiful sources of bioactive peptides that can be harnessed for the benefits of human health.

Thermoase-hydrolysed pigeon pea protein and its membrane fractions possess in vitro bioactive properties (antioxidative, antihypertensive, and antidiabetic).

Enzymatic hydrolysis can liberate bioactive peptides from protein materials, thus, pigeon pea was hydrolysed using thermoase. Crude hydrolysate (PPHT) was subjected to ultrafiltration using different molecular weight cutoffs to collect <1, 1-3, 3-5, 5-10, and >10kDa peptide fractions. Fractions were analysed for in vitro antioxidative, antihypertensive, and antidiabetic properties. The peptide fractions had stronger DPPH• scavenging and renin inhibition when compared to PPHT. In contrast, ACE inhibition was stronger for the PPHT and <1kDa peptide fraction while activity decreased as peptide size increased. The <1kDa peptide also showed significantly stronger ferric reducing antioxidant power, OH• scavenging and inhibition of linoleic acid oxidation when compared to PPHT. α-amylase and α-glucosidase were inhibited by all the peptide fractions, though the 3-5 and >10kDa had higher values. We conclude that the PPHT and peptide fractions could serve as potential ingredients to formulate antihypertensive and antidiabetic functional foods and nutraceuticals. PRACTICAL APPLICATIONS: Oxidative stress promotes the generation of free radicals, which have a significant impact in the pathogenesis of human chronic diseases such as cardiovascular impairment, cancer, and diabetes. Peptides generated from enzymatic hydrolysis of proteins have been identified to impart beneficial health effects. In this work, we showed that a thermoase digest of pigeon pea protein as well as the fractionated peptides had strong antioxidant properties in addition to exhibiting inhibitory activities against renin and angiotensin converting enzyme, the main therapeutic targets for antihypertensive agents. The peptide products also inhibited α-amylase and α-glucosidase activities, providing potential ingredients that can be used to formulate antidiabetic functional foods.

Increased Production of the Value-Added Biopolymers Poly(R-3-Hydroxyalkanoate) and Poly(γ-Glutamic Acid) From Hydrolyzed Paper Recycling Waste Fines.

Reject fines, a waste stream of short lignocellulosic fibers produced from paper linerboard recycling, are a cellulose-rich paper mill byproduct that can be hydrolyzed enzymatically into fermentable sugars. In this study, the use of hydrolyzed reject fines as a carbon source for bacterial biosynthesis of poly(R-3-hydroxyalkanoate) (PHA) and poly(γ-glutamic acid) (PGA) was investigated. Recombinant Escherichia coli harboring PHA biosynthesis genes were cultivated with purified sugars or crude hydrolysate to produce both poly(R-3-hydroxybutyrate) (PHB) homopolymer and medium chain length-containing copolymer (PHB-co-MCL). Wild-type Bacillus licheniformis WX-02 were cultivated with crude hydrolysate to produce PGA. Both PHB and short chain-length-co-medium chain-length (SCL-co-MCL) PHA yields from crude hydrolysate were a 2-fold improvement over purified sugars, and the MCL monomer fraction was decreased slightly in copolymers produced from crude hydrolysate. PGA yield from crude hydrolysate was similarly increased 2-fold. The results suggest that sugars from hydrolyzed reject fines are a viable carbon source for PHA and PGA biosynthesis. The use of crude hydrolysate is not only possible but beneficial for biopolymer production, eliminating the need for costly separation and purification techniques. This study demonstrates the potential to divert a lignocellulosic waste stream into valuable biomaterials, mitigating the environmental impacts of solid waste disposal.

In Silico and In Vitro Assessment of Portuguese Oyster (Crassostrea angulata) Proteins as Precursor of Bioactive Peptides.

In this study, the potential bioactivities of Portuguese oyster (Crassostrea angulata) proteins were predicted through in silico analyses and confirmed by in vitro tests. C. angulata proteins were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by proteomics techniques. Hydrolysis simulation by BIOPEP-UWM database revealed that pepsin (pH > 2) can theoretically release greatest amount of bioactive peptides from C. angulata proteins, predominantly angiotensin I-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory peptides, followed by stem bromelain and papain. Hydrolysates produced by pepsin, bromelain and papain have shown ACE and DPP-IV inhibitory activities in vitro, with pepsin hydrolysate (PEH) having the strongest activity of 78.18% and 44.34% at 2 mg/mL, respectively. Bioactivity assays of PEH fractions showed that low molecular weight (MW) fractions possessed stronger inhibitory activity than crude hydrolysate. Overall, in vitro analysis results corresponded with in silico predictions. Current findings suggest that in silico analysis is a rapid method to predict bioactive peptides in food proteins and determine suitable enzymes for hydrolysis. Moreover, C. angulata proteins can be a potential source of peptides with pharmaceutical and nutraceutical application.

Development of a Circular Oriented Bioprocess for Microbial Oil Production Using Diversified Mixed Confectionery Side-Streams.

Diversified mixed confectionery waste streams were utilized in a two-stage bioprocess to formulate a nutrient-rich fermentation media for microbial oil production. Solid-state fermentation was conducted for the production of crude enzyme consortia to be subsequently applied in hydrolytic reactions to break down starch, disaccharides, and proteins into monosaccharides, amino acids, and peptides. Crude hydrolysates were evaluated in bioconversion processes using the red yeast Rhodosporidium toruloides DSM 4444 both in batch and fed-batch mode. Under nitrogen-limiting conditions, during fed-batch cultures, the concentration of microbial lipids reached 16.6–17 g·L−1 with the intracellular content being more than 40% (w/w) in both hydrolysates applied. R. toruloides was able to metabolize mixed carbon sources without catabolite repression. The fatty acid profile of the produced lipids was altered based on the substrate employed in the bioconversion process. Microbial lipids were rich in polyunsaturated fatty acids, with oleic acid being the major fatty acid (61.7%, w/w). This study showed that mixed food side-streams could be valorized for the production of microbial oil with high unsaturation degree, pointing towards the potential to produce tailor-made lipids for specific food applications. Likewise, the proposed process conforms unequivocally to the principles of the circular economy, as the entire quantity of confectionery by-products are implemented to generate added-value compounds that will find applications in the same original industry, thus closing the loop.