Enzymatic Hydrolysis Research Articles

Systematic Development of Peptide-Based Biostimulants from Whey Protein Hydrolysates

Due to global warming, agricultural production systems are exposed to increasing abiotic stresses, which threaten an economically and environmentally sustainable food production. Innovative environmentally friendly solutions are needed to cope with climate-related risks and to reduce the use of high amounts of synthetic agrochemicals. A promising solution to foster sustainable agriculture involves the use of biostimulants. This strategy, however, is not adopted by the industry to a great degree due to the scarce number of reproducible effects observed in published studies as well as the lack of fundamental knowledge about their mode of action. Biostimulants are substances that positively impact plant physiology by enhancing growth, improving fruit quality, and increasing stress resilience. Among these, protein hydrolysates stand out as a particularly promising category. However, their precise mechanisms of action and the optimal conditions for their application are still not fully understood. This project aims to develop peptide-based biostimulants in a reproducible manner, ensuring their availability for use in plant model systems under strictly controlled conditions. To achieve this, various protein hydrolysates will be produced through the enzymatic hydrolysis of whey, a by-product of cheese production. These hydrolysates will be employed in subsequent studies within plant model systems.

Enzymatic conversion of wood materials from the pulp and paper industry

The reactivity during enzymatic hydrolysis of 8 industrially produced samples of pulps and semi-chemical pulps by enzyme preparations of glycosyl hydrolases B151 and F10 produced by a strain of ascomycete fungus Penicillium verruculosum has been determined. It is shown for the first time that among fibrous pulps available on the market of pulp and paper industry in Russia, the highest level of yield of glucose from the initial wood during biocatalysis using cellulases and hemicellulases is characteristic of semi-chemical pulps obtained after cooking of hardwood with green liquor. A high degree of enzymatic conversion of softwood bleached kraft pulp has been established, which in combination with the possibility of obtaining modified polysaccharide materials from non-hydrolysable residue makes this cellulosic substrate the most promising for the development of biological processes at pulp and paper industries. It is shown that drying of pulp negatively affects the efficiency of cellulose hydrolysis, while mechanical milling improves the performance of the enzymatic saccharification process.

Proton-Mediated Dynamic Nestling of DNA Payloads Within Size-Matched MOFs Nanochannels for Smart Intracellular Delivery.

With sequence-programmable biological functions and excellent biocompatibility, synthetic functional DNA holds great promise for various biological applications. However, it remains a challenge to simultaneously retain their biological functions while protecting these fragile oligonucleotides from the degradation by nucleases abundant in biological circumstances. Herein, a smart delivery system for functional DNA payloads is developed based on proton-mediated dynamic nestling of cytosine-rich DNA moieties within the precisely size-matched nanochannels of highly crystalline metal-organic frameworks (MOFs): At neutral pH, cytosine-rich DNA strands exhibit a flexible single-stranded state and can be accommodated by MOFs nanochannels with a size of ca. 2.0nm; while at acidic conditions, the protonation of cytosine-rich strands weakens their interaction with the nanochannels, and the tendency to form four-strandedstructures drives these DNA strands out of the nanochannels. Results confirm the successful protection of DNA payloads from enzymatic hydrolysis by the MOFs nanochannels, and the delicate coupling of the endocytosis processes and the proton-responsive Cytosine-rich DNA/MOFs systems realized the efficient intracellular delivery of DNA payloads. Furthermore, with a complementary sequence to the telomere overhangs, direct imaging of telomeres and the nucleus is successfully achieved with the proton-mediated DNA/MOFs system.

Enhancing Lignin-Carbohydrate Complexes Production and Properties With Machine Learning.

Lignin-carbohydrate complexes (LCCs) present a unique opportunity for harnessing the synergy between lignin and carbohydrates for high-value product development. However, producing LCCs in high yields remains a significant challenge. In this study, we address this challenge with a novel approach for the targeted production of LCCs. We optimized the AquaSolv Omni (AqSO) biorefinery for the synthesis of LCCs with high carbohydrate content (up to 60/100 Ar) and high yields (up to 15 wt %) by employing machine learning (ML). Our method significantly improves the yield of LCCs compared to conventional procedures, such as ball milling and enzymatic hydrolysis. The ML approach was pivotal in tuning the biorefinery to achieve the best performance with a limited number of experimental trials. Specifically, we utilized Bayesian Optimization to iteratively gather data and examine the effects of key processing conditions-temperature, process severity, and liquid-to-solid ratio-on yield and carbohydrate content. Through Pareto front analysis, we identified optimal trade-offs between LCC yield and carbohydrate content, discovering extensive regions of processing conditions that produce LCCs with yields of 8-15 wt % and carbohydrate contents ranging from 10-40/100 Ar. To assess the potential of these LCCs for high-value applications, we measured their glass transition temperature (Tg), surface tension, and antioxidant activity. Notably, we found that LCCs with high carbohydrate content generally exhibit low Tg and surface tension. Our biorefinery concept, augmented by ML-guided optimization, represents a significant step toward scalable production of LCCs with tailored properties.

Ulva clathrata Enzymatic Hydrolysate Protects Wheat from Salt–Alkaline Stress by Regulating ABA Biosynthesis

Ulva clathrata Enzymatic Hydrolysate Protects Wheat from Salt–Alkaline Stress by Regulating ABA Biosynthesis

Insight into Antifungal Metabolites from Bacillus stercoris 92p Against Banana Cordana Leaf Spot Caused by Neocordana musae

Banana crop ranks among the most crucial fruit and food crops in tropical and subtropical areas. Despite advancements in production technology, diseases such as cordana leaf spot, caused by Neocordana musae, remain a significant challenge, reducing productivity and quality. Traditional chemical controls are becoming less effective due to the development of resistance in target pathogens, which pose significant environmental and health concerns. Consequently, there is growing attention toward the development of biocontrol strategies. Here, we identified a new bacterial strain, Bacillus stercoris 92p, from the rhizosphere soil of banana. We evaluated its ability to suppress the growth of N. musae and other fungal pathogens that cause leaf spot disease in bananas. The inhibitory effect of B. stercoris 92p were checked using dual culture assays, microscopic observations, and pot experiments. Furthermore, the biocontrol mechanisms were investigated using whole-genome sequencing and biochemical analyses. The results showed that B. stercoris 92p exhibited significant antifungal activity against N. musae and other fungal pathogens, with inhibition rates exceeding 70%. Microscopic examination revealed significant morphological alterations in the hyphae and conidia of the tested pathogens. In pot experiments, B. stercoris 92p effectively reduced the severity of cordana leaf spot, achieving a biocontrol efficacy of 61.55%. Genomic analysis and biochemical tests indicated that B. stercoris 92p produces various antifungal compounds, including lipopeptides (fengycins and surfactins), hydrolytic enzymes (proteases and amylases), and phosphate-solubilizing metabolites. In conclusion, the study highlights that B. stercoris could potentially be used as a potential biological control agent against cordana leaf spot.

Mechanism of protein component addition on digestive properties of starch in fermented barley

Previous studies have found that removing protein from fermented barley flour promotes starch hydrolysis. In this paper, we investigate how the addition of fermented barley proteins to fermented starch affects starch digestive properties and explore the underlying mechanisms. The results show that the starch-protein complex (with a mass ratio of 1:0.35) forms larger protein aggregates during the co-gelatinization of starch and protein, which limits the swelling of starch and thus inhibits starch digestion. Analysis of the peak positions in the infrared spectrograms revealed that the absorption peaks of the starch-protein complexes shifted from 3313.65 cm−1 to 3302.49 cm−1. The addition of sodium dodecyl sulfate (SDS) to some extent lowered the storage modulus (G′) and loss modulus (G″) of the mixed system. The proteins confer a stronger resistance to enzymatic hydrolysis on the starch through the formation of hydrogen bonds and participation in hydrophobic interactions. The protein inhibits the activity of α-amylase, with inhibition as high as 77.32% at a protein concentration of 80 mg/mL and a semi-inhibitory concentration of 35.22 mg/mL. Thus, the addition of protein attenuates starch digestion. These findings elucidate the mechanism of protein effects on starch digestion properties during fermented barley digestion and provide a theoretical basis for the subsequent preparation of low-glycemic starch foods.

An efficient measure for the isolation of chenodeoxycholic acid from chicken biles using enzyme-assisted extraction and macroporous resins refining

Chicken bile is a by-product of chicken processing, rich in chenodeoxycholic acid (CDCA), an active pharmaceutical raw material. In this study, a green measure for the extraction and purification of CDCA from chicken biles by enzymatic hydrolysis and macroporous resins refining was established. For the assisted extraction of CDCA, the active bile salt hydrolase (BSH) from Bifidobacterium was heterologously expressed and applied, its activities on GCDCA and TCDCA were 4.96 ± 0.32 U/mg and 3.07 ± 0.031 U/mg and optimal catalytic conditions for the extraction of CDCA were determined as 0.04 g/g of the enzyme dosage, pH 5.0 and 38 °C. Through validation of the conditions, the yield of CDCA was up to 5.32 %, which was equivalent to that by saponification method. In order to further refine CDCA from the extract obtained by enzyme-assisted extraction, a more preferable resin, AB-8 was selected for the purification of CDCA, which had a good adsorption capacity of 61.06 ± 0.57 mg/g for CDCA. Besides, the obtained CDCA extract was purified through AB-8 resin, the purity of CDCA was improved from 51.7 % to 91.4 % and the recovery yield of CDCA was 87.8 %. The advantages of energy conservation, time saving, economy and environmental friendliness make the measure using enzyme-assisted extraction and macroporous resins refining a promising candidate for isolation of CDCA from chicken bile.

Antihypertensive peptides from hydrolyzed proteins of Pleurotus spp.: production, in vitro digestion and identification

This study aimed to investigate in vitro antihypertensive property of protein hydrolysates from Pleurotus spp. mushrooms obtained through enzymatic hydrolysis. FlavourzymeTM 500L, AlcalaseTM 2.4L and NeutraseTM 0.8L were used alone and in combination using an experimental design of mixtures. Antihypertensive activity was determined by angiotensin-converting enzyme (ACE) inhibition and the simulated in vitro digestion was performed according to the INFOGEST protocol. Results showed that most of the protein hydrolysates obtained displayed higher ACE inhibitory activity than non-hydrolyzed protein, ranging from 15.76% and 50.87% inhibition. The highest ACE inhibition and the lowest TCA soluble protein content (52.09%) were detected for the protein hydrolysates obtained by using the binary mixture of FlavourzymeTM 500L and AlcalaseTM 2.4L in equal proportions. Hydrolysis kinetics showed no significant difference in ACE inhibitory activity between 20 and 120minutes of enzymatic reaction. The fraction with molecular weight between 3 and 5kDa obtained after ultrafiltration showed the most contribution for ACE inhibitory activity (47% inhibition). After simulated digestion, the hydrolysates maintained a significant ACE inhibition capacity, indicating the resistance of the peptides to the action of gastrointestinal enzymes. Through proteomic analysis, 26 peptides were identified and the sequences LPILP, IPLLP, PLLPQ and VIQYDPPQ were considered potentially antihypertensive.

Collagen (peptide) extracted from sturgeon swim bladder: Physicochemical characterization and protective effects on cyclophosphamide-induced premature ovarian failure in mice

Premature ovarian failure (POF) is a common female reproductive disorder, with oxidative damage playing a significant role in its development. Studies had demonstrated the beneficial antioxidant, anti-aging, and other biological properties of swim bladder collagen (peptide). In this study, acid extraction, water extraction, and enzymatic hydrolysis methods were used to extract collagen (peptide) from sturgeon (Acipenser sinensis) swim bladder, all of which exhibited significant antioxidant activity. Moreover, the effects observed in POF mice were enhanced, including an increasing in the number of growing ovarian follicles, regulation of serum hormone levels, and alterations in signaling pathways, as evidenced by the up-regulation of Phosphatidylinositol 3-kinase/Protein Kinase B (PI3K/Akt) and B cell lymphoma-2/ Bcl-associated x protein (Bcl-2/Bax) pathways, alongside the down-regulation of the mitogen-activated protein kinases (MAPK) signaling pathway. Sturgeon swim bladder collagen (peptide) could protect against cyclophosphamide-induced POF in mice, which could be very beneficial in the future advancement of health products.

Application of High-pressure Carbon Dioxide in Japanese Anchovy Waste Recycling by Enzymatic Hydrolysis.

Anchovy waste, a protein resource with high nutritional value and potential for recycling with a relatively high economic effect, is essential for the Sustainable Development Goals of the United Nations. Preventing microbial contamination during the recycling process, through enzymatic hydrolysis, ensures the safety of recycled products. High-pressure carbon dioxide is a novel non-thermal decontamination technology, which inactivates cells by breaking their membranes. Here, we selected 40 °C_5.0 MPa and 50 °C_1.0 MPa treatment conditions for effectively decontaminating anchovy samples during the hydrolysis process. Next Generation Sequencing and real-time PCR experiments showed that a microbial growth promotion stage existed at the beginning of 40 °C_5.0 MPa, which may threaten hydrolysates, as some microbial genera were detected from the metabolites produced. Treatment at 50 °C_1.0 MPa ensured a high safety level for hydrolysates but this is limiting for various enzymatic hydrolysis processes. Orientaaze OP was selected as an additional enzyme with the highest hydrolysis efficiency under 40 and 50 °C among 10 different industrial proteases. Compared with control samples without high-pressure carbon dioxide treatment, 40 °C_5.0 MPa and 50 °C_1.0 MPa treated samples presented higher total amino acid concentrations by ultra-high-performance liquid chromatography. Hence, there was an increased enzyme activity by 40 °C_5.0 MPa and 50 °C_1.0 MPa treatments in endogenous or additional proteases hydrolytic processes. Despite the need for more future studies to be conducted, this research still provides essential information and instruction for industrial enzymatic hydrolysis applications on anchovy waste recycling.

Modification of regenerated cellulose fibres by cork-derived suberin and the cutin fraction from grape skins

Regenerated cellulose fibres from dissolving pulp are a versatile alternative to cotton fibres on the path to the sustainable textile industry. In this study, cellulose fibres obtained by the Ioncell-F® process (Ioncell fibres) were modified by adding 10 % (w/w) of suberin compounds isolated from cork (SUB) or a cutin fraction from grape skins (CUT) in the spinning dope. Although both SUB and CUT modified fibres revealed higher hydrophobicity than unmodified fibres, fibres doped with CUT showed better waterproof performance than those doped with SUB. This was explained by the better retention of CUT than SUB on the regenerated fibres and by the higher hydrophobicity of CUT. Differences in the strength properties of Ioncell fibres obtained by pilot-scale dry-jet wet spinning were related to their physical structure, whereas dirt repellence and susceptibility to enzymatic hydrolysis depended on the occurrence and amounts of retained CUT or SUB.

Enhancement of foaming performance of hempseed protein by limited enzymatic hydrolysis: From the viewpoint of the structural and interfacial rheological attributes

Effects of limited enzymolysis by Alcalase and Protamex on the foaming performance of hempseed protein (HPI) and its correlation with structural and interfacial rheological characteristics were investigated. Proteolysis induced a conformational shift from α-helix and β-sheet to random coil, indicating enhanced molecular flexibility. The surface hydrophobicity of Alcalase hydrolysates encountered an initial increase (5 min) followed by a sharp drop with prolonged hydrolysis, whereas Protamex showed minimal effects. Both proteases reduced total sulfhydryl content and caused a redshift in intrinsic fluorescence, particularly Alcalase. The specific cleavage pattern of Alcalase generated peptides with pronouncedly higher solubility (up to 47.0 %) relative to Protamex. The flexible conformation and increased solubility induced by moderate proteolysis, notably with Alcalase, facilitated viscoelastic interfacial membranes with consolidated intermolecular interactions, consequently contributing to more homogeneous and smaller bubble structures. Optimal foaming capacity (95.0 %) and foam stability (90.9 %) were achieved with Alcalase for 20 min and 5 min, respectively.

Implementation of Sensitive Method for Determination of Benzophenone and Camphor UV Filters in Human Urine

The level of the human body’s burden of benzophenone and camphor ultraviolet (UV) filters can be estimated from their urinary levels. The present study describes the implementations and validation of the sensitive analytical method for the analysis of seven benzophenone and two camphor UV filters in urine. Sample preparation includes overnight enzymatic hydrolysis and ethyl acetate extraction followed by purification by dispersive solid-phase extraction using a sorbent Z-Sep. For the analysis, ultra-high performance liquid chromatography coupled with tandem mass spectrometry was used. Validation was performed using a Standard Reference Material® 3673 and an artificially contaminated urine sample. Target analyte recoveries ranged from 79–113% with repeatability expressed as a relative standard deviation of 2–15%. The limits of quantification were between 0.001 and 0.100 ng/mL in urine. This method was subsequently applied to examine the urine samples collected from Czech women. The analytes benzophenone-1 and 4-hydroxy-benzophenone were the most common analytes present in 100% of the samples, whereas benzophenone-3 was quantified in only 90% of the urine samples. The other four determined benzophenone derivatives were quantified in ≤33% of the samples. The derivatives of camphor were not detected in any samples. This method could be applied in biomonitoring studies.

Comparative analysis of different pretreatments and hydrolysis conditions for the generation of taste-related substances in pork liver hydrolyzates

In recent years, the increase in meat industry production supposes a negative impact in the environment. Revaluation of meat by-products is essential to mitigate this impact and enzymatic hydrolysis of proteins, aided by pretreatment techniques like ultrasound, offers a promising approach to produce hydrolyzates with potential as functional ingredients. This study evaluates optimal conditions for enzymatic hydrolysis of pork liver, focusing on ultrasound and thermal pretreatment, and optimizing enzyme selection to enhance the production of bioactive peptides and taste-related compounds.Results demonstrate that probe ultrasound pretreatment significantly increases umami amino acids and the release of taste-related amino acids in cooked Alcalase hydrolyzates. This effect is further amplified in raw liver Alcalase hydrolyzates. Additionally, ultrasound pretreatment of Flavourzyme hydrolyzates improves both the release of taste-related amino acids and antioxidant activity. Overall, pork liver shows great potential as a raw material for producing hydrolyzates with potential applications as functional ingredients.

Maleic Acid-Butanol Pretreatment to Enhance Cellulose Accessibility for Enzymatic Hydrolysis and Ethanol Production from Oil Palm Empty Fruit Bunch

Maleic Acid-Butanol Pretreatment to Enhance Cellulose Accessibility for Enzymatic Hydrolysis and Ethanol Production from Oil Palm Empty Fruit Bunch

Elucidating the impact of dual modification with hydroxypropylation and enzymatic hydrolysis on physicochemical properties of corn and sago starch

Enzymatic hydrolysis is one of the most commonly used techniques in starch modification. However, the performance of enzymatic hydrolysis has been restricted by the inherent properties of native starch. In this study, hydroxypropylation with two different concentration levels of propylene oxide (10% and 20%) was carried out on corn and sago starches. The effect of hydroxypropylation on the physico-chemical properties of the enzyme-hydrolyzed starch was investigated. The results showed that the starches treated with 20% propylene oxide had higher molar substitution and dextrose equivalent with greater effects. Scanning electron microscope (SEM) revealed that hydoxypropylation had developed pores and pits on the granular surfaces and subsequently enhanced enzyme penetration. Hydroxypropylation showed no changes in the crystalline pattern, yet a decrease in the amylose content of both corn and sago starches, respectively. Other than that, hydroxypropylation decreased the pasting properties and retrogradation tendency but increased the swelling power of enzymehydrolyzed starch significantly. Overall, this work suggested that hydoxypropylation pretreatment could improve the efficiency of subsequent enzyme hydrolysis and produce modified starch with great potential in food processing applications.

Oral Intake of Collagen and Collagen Hydrolysate From Takifugu bimaculatus Attenuates Ultraviolet‐Induced Skin Photoaging in Mice

ABSTRACTWith the rapid emergence of pufferfish aquaculture and processing industries, fish skin is underutilized as a byproduct of processing, leading to resource waste. In this study, Takifugu bimaculatus skin collagen (TBSC) was extracted by acetic acid solubilization and its physicochemical properties were analyzed. The effects of TBSC and the TBSC hydrolysate (TBSCH) on ultraviolet (UV) irradiation‐induced photoaging were investigated using a mouse model. The purity of TBSC was 90.02%. Electrophoresis and Fourier infrared spectroscopy characterization of TBSC showed that the type of collagen in TBSC was typical standard type I. The degree of hydrolysis was selected to optimize the hydrolysis conditions for TBSC. The papain enzyme dosage, temperature, pH, and hydrolysis duration of 51,000 U/g, 48.03°C, 5.35, and 4 h have been demonstrated to be the optimum hydrolysis conditions for TBSCH. Oral administration of either TBSC or TBSCH ameliorated UV‐induced skin erythema and hyperkeratosis. TBSC and TBSCH treatment increased collagen content and had an inhibitory effect on matrix metalloproteinase (MMP)‐2 and MMP‐3 expression, whereas MMP‐9 expression was significantly reversed only in the TBSCH‐treated groups. The expression of the c‐Jun protein was much lower in these groups, suggesting that TBSCH had a greater alleviating effect on collagen degradation and extracellular matrix breakdown. Therefore, it is proposed that TBSCH has the potential to be used as a nutricosmetic agent with protective attributes against UV‐induced skin damage and concurrent collagen depletion.

Purification and characterization of a protease produced by submerged fermentation: Ultrasound-enhanced collagenolytic protease from Streptomyces parvulus

Proteases are a large group of enzymes in high demand due to their wide and different biotechnological applications mainly in the biomedical field. Ultrasound (US) has been used successfully in several Bioprocesses in biotechnology, such as in the upregulation of enzymatic hydrolysis (biocatalysis). The objective of this work was to purify an enzyme from Streptomyces parvulus and to characterize it through physic-chemical applications including ultrasound effect. The purified protease has a molecular weight of 78.0 KDa, a yield of 31 % and 11.8-fold, it was stable between pH 4–9, optimum pH at 7.5, temperature of 0–45 °C, and showed optimum temperature at 45 °C, exhibited enhanced activity with Ca2+ and Mg2+, and was inhibited by PMSF. US in the treatment or pre-treatment of enzymatic reactions showed to be favorable and increase the activity around 85 % for the optimum temperature 45 °C. Also, in circular dichroism spectra it was shown a significant change in enzyme structure under US effect enhancing the real activity. Besides, the US improved the enzyme reactions for all assays. The purified enzyme was successfully immobilized in chitosan film. Thus, the present work demonstrated the promising results of a protease with collagenolytic activity in the field of Biotechnology by proving the positive effect induced by ultrasound.

Composite enzymatic hydrolysis for producing Harlikar apple juice with increased dietary fiber content and antioxidant capacity

The Harlikar apple is a newly developed high-quality apple variety that is rich in nutrients and has a unique flavor, making it highly popular among customers. However, current researches on Harlikar apples are limited to cultivation and storage, with little attention paid to the production process. Additionally, the optimization of enzymatic conditions has rarely focused on dietary fiber, resulting in the waste of this nutrient in the apple pomace. In this study, cellulase, pectinase, xylanase and α-amylase were utilized as a composite enzyme to produce Harlikar apple juice with increased dietary fiber content and antioxidant capacity. The optimal enzymatic hydrolysis conditions were determined based on juice yield, clarity, total phenolic content (TPC) and dietary fiber content as follows: cellulase 0.1% (w/w), pectinase 0.04% (w/w), xylanase 0.1% (w/w), α-amylase 0.04% (w/w), pH 5, temperature 45 °C and reaction time 4 h. Compared with the apple juice without enzymatic hydrolysis, the juice yield, clarity, TPC and dietary fiber content increased form 37.50%, 4.45%, 91.04 mg gallic acid equivalents (GAE)/100 mL and 0.1343 g/100g to 85.49%, 28.77%, 220.36 mg GAE/100 mL and 0.7585 g/100g, respectively. The antioxidant properties were significantly enhanced, and the sugar and acid contents were also increased to varying degrees.