Original Hydrolysate Research Articles

Maillard reaction products of hydrolyzed Copyinds comatus protein with various sugar-amino acid combinations: Focusing on their meaty aroma notes and salt reduction abilities

Maillard reaction products (MRPs) of mushroom protein have been shown to present unique flavor properties, but how additional reducing sugar and amino acids manipulate the generation of meaty flavor and the taste traits of MRPs are still open questions. In this study, the Copyinds comatus protein hydrolysates with different reducing sugars (D-ribose and D-xylose) and amino acids (cysteine, methionine, proline, and glutamic acid) were thermally treated. The flavor properties, volatile compounds and taste profiles of the resulting products were comprehensively evaluated using multi-scale flavor analyses techniques. Though the E-nose and E-tongue revealed enriched volatile profiles and taste traits of MRPs than the original hydrolysate, MRPs derived from various sugar-amino acid combinations cannot be well distinguished according to the PCA score plots. The fingerprints by GC-IMS revealed amino acid-specific compounds, including methylpyrazine, hexanol, and isopropyl acetate for methionine-participated MRPs, and ethyl propanoate, cyclohexanone, and 2-butanone for MRPs derived from cysteine. Quantitative results further demonstrated that the methionine-involved MRPs contained significantly higher amounts of sulfur- and nitrogen-containing VOCs than other MRPs, most of which were associated with meat-like odors. Sensory evaluation confirmed that the species of amino acid and reducing sugars made differences in the taste traits, and MRPs prepared from xylose-glutamic acid and xylose-methionine showed outstanding advantages of saltiness and umami taste enhancement. Overall, Copyinds comatus MRPs with methionine are efficient ingredients for the production of meat flavoring, with capacities to compensate for reduced sensory properties of low-sodium food.

Enhancing the Amino Acid and Reducing the Metal Ions Contents in the Hydrolysate Resulting from Hydrothermal Carbonization of Chicken Feather Waste by Chemical Phosphorylation

Chemical phosphorylation of hydrolysate resulting from hydrothermal carbonization of chicken feather waste was performed to enhance the amino acids and reduce the metal ions content. The aim of this research is to improve the functional properties of chicken feathers hydrolysate without impairing the nutritional availability thereof with the cheapest chemical method by phosphorylation. Phosphorylated hydrolysate can function as animal feed and fertilizer. The hydrolysate of chicken feathers was obtained by hydrothermal carbonization in an alkaline condition using a CaO and KOH catalyst, by the ratio of water:dry matter of chicken feathers is 5:1, at 9–10 atm pressure, and in a temperature of 190–200 °C during 3 h. Phosphorylation has been carried out by reacting the hydrolysate with H3PO4 85% in pH of 5, 6, 7 and using the original hydrolysate as control. The sample that has been prepared was characterized and semi-quantitative analyzed by HPLC and AAS. The phosphorylation results showed that the total maximum protein of soluble protein, their minimum metal ions, and anion in soluble protein was obtained at pH 7, while the higher the pH, the lower the liquid protein that was obtained.

Production and characterisation of PHAs by pure culture using protein hydrolysates as sole carbon source

Protein hydrolysates obtained from discarded biomass can be further upgraded into high market value products, in the optic of a circular bioeconomy. In this work, residues from the cultivation of alfalfa, soybean and rice, and bovine wet blue shavings were fermented with mixed microbial cultures obtaining high concentrations of volatile fatty acid (up to 50 times compared to the original hydrolysate), mainly butyric and acetic acid. This rich medium was used for growing the bacterium Thauera sp., a known producer of polyhydroxyalkanoates (PHAs), biodegradable polymers with potential to replace petrol-based plastics. The overall process resulted in the production of 1.4 gPHAs/L, with a conversion rate of 32% for the alfalfa hydrolysates when considering the COD given by the initial VFAs. The obtained biopolymer was poly(3-hydroxybutyrate-co-3-hydroxyvalerate), as confirmed by the presence of characteristic peaks and by the melting temperatures and thermo-oxidative degradation in the expected range; the polymer has a high degree of purity, being without inorganic residues. This work showed the feasibility of a process aimed at the valorisation of protein hydrolysates into high-market value products such as bioplastics. • Leftover animal & vegetal biomass was valorised into COD-rich protein hydrolysates. • High VFA production was obtained by fermentation of four types of hydrolysates. • Growth with Thauera allowed the production of the biodegradable polyesters PHAs. • The Alfalfa hydrolysate produced up to 1,4 g/L of PHBV with a high degree of purity. • Hydrolysates were upgraded into high-value products with a process yield of 32%

Novel Natural Angiotensin Converting Enzyme (ACE)-Inhibitory Peptides Derived from Sea Cucumber-Modified Hydrolysates by Adding Exogenous Proline and a Study of Their Structure⁻Activity Relationship.

Natural angiotensin converting enzyme (ACE)-inhibitory peptides, which are derived from marine products, are useful as antihypertensive drugs. Nevertheless, the activities of these natural peptides are relatively low, which limits their applications. The aim of this study was to prepare efficient ACE-inhibitory peptides from sea cucumber-modified hydrolysates by adding exogenous proline according to a facile plastein reaction. When 40% proline (w/w, proline/free amino groups) was added, the modified hydrolysates exhibited higher ACE-inhibitory activity than the original hydrolysates. Among the modified hydrolysates, two novel efficient ACE-inhibitory peptides, which are namely PNVA and PNLG, were purified and identified by a sequential approach combining a sephadex G-15 gel column, reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), before we conducted confirmatory studies with synthetic peptides. The ACE-inhibitory activity assay showed that PNVA and PNLG exhibited lower IC50 values of 8.18 ± 0.24 and 13.16 ± 0.39 μM than their corresponding truncated analogs (NVA and NLG), respectively. Molecular docking showed that PNVA and PNLG formed a larger number of hydrogen bonds with ACE than NVA and NLG, while the proline at the N-terminal of peptides can affect the orientation of the binding site of ACE. The method developed in this study may potentially be applied to prepare efficient ACE-inhibitory peptides, which may play a key role in hypertension management.

Protection effect of donkey hide gelatin hydrolysates on UVB-induced photoaging of human skin fibroblasts

Abstract UVB irradiation is a potent photoaging factor and leads to the formation of skin wrinkles. The objective of this study was to investigate the mechanisms of UVB photoaging inhibition of low molecular weight peptides derived from donkey hide gelatin hydrolysates (LDGH) on human skin fibroblasts. The donkey hide was hydrolyzed using proteases, and the LDGH exhibited higher antioxidant activity than the original hydrolysate and inhibited collagenase and elastase activities. We also found that LDGH increases viability of Hs68 cells after UVB irradiation at 100 mJ/cm2. Moreover, LDGH prevented the decrease in procollagen type I levels caused by exposure to UVB irradiation in Hs68 cells and reduced the up-regulated phosphorylation of p38, ERK, and JNK in the mitogen-activated protein kinase (MAPK) signaling pathway. These findings suggest that LDGH increases synthesis of procollagen type I by decreasing the phosphorylation of MAPK, therefore, LDGH may be useful as an effective anti-photoaging agent.

Bioethanol production from detoxified hydrolysate and the characterization of oxalic acid pretreated Eucalyptus (Eucalyptus globulus) biomass

In this study, different detoxification treatments (XAD-4 resin adsorption [XAD] and electrodialysis) on oxalic acid pretreated hydrolysate were evaluated. Two different detoxification processes were applied to remove fermentation inhibitors from the oxalic acid pretreated hydrolysate of eucalyptus. The hydrolysates contain sugars (glucose, xylose, and arabinose) and inhibitors. Acetic acid was the main inhibitor in the original hydrolysate, and more total phenolic compounds were generated than hydroxymethyl furfural and furfural. The acetic acid was removed 100% by the electrodialysis process, and total phenolic compounds and furfural were effectively removed from the hydrolysate by XAD. Based on the process order, the combined electrodialysis-XAD process was more effective for ethanol fermentation compared to the combined XAD-electrodialysis process. Ethanol fermentation was successfully performed using the electrodialysis-XAD-treated hydrolysate and achieved a production of 7.83g/L of ethanol, corresponding to an ethanol yield of 0.40g/g within 48h. Fourier transform infrared, X-ray diffractometer, and scanning electron microscopy were used to investigate the characteristics of the raw material and pretreated biomass. The results showed that most of the hemicellulose was degraded during oxalic acid pretreatment. Therefore, the crystallinity value increased from 49.79% to 56.93%. The surface of the raw material was smooth, plump, and compact, whereas the pretreated biomass was more fragmented.

목질계 바이오매스로부터 생산된 바이오에탄올 투과증발 과정에서 발생한 투과증발 잔류물의 항산화 활성

In this study, we produced bioethanol from the original hydrolysate obtained during oxalic acid pretreatment of lignocellulosic biomass. The bioethanol was separated and concentrated by pervaporation and the residue after pervaporation was evaluated for its antioxidant activity. Xylose () was the major product in the original hydrolysate. The original hydrolysate contained acetic acid, furfural and total phenolic compounds (TPC) as fermentation inhibitors. Acetic acid was removed by electrodialysis (ED), and of bioethanol was produced from ED-treated hydrolysate. The TPC of ethyl acetate extracts from the residue obtained (OA-E) during pervaporation was 86.81 mg/100 g (extract). The values of DPPH and ABTS radical scavenging activities, and reducing power of OA-E were , , and , respectively. Sugar degradation products and the phenolic compounds in OA-E were determined by GC-MS.

English

The enzymatic hydrolysate of freshwater clam (Corbicula fluminea) extract was prepared using commercially available proteases. The antioxidant activity of the hydrolysate was evaluated by reducing power and 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid free radical decolorization assays. The hydrolysate, especially its fraction purified by gel filtration, had a good antioxidant activity. The Trolox equivalent antioxidant capacity of the fraction with the highest radical scavenging ability reached 158.04 ± 2.43 μg/mg, which was sevenfold higher than that of the original hydrolysate. Molecular weight distribution analysis revealed that peptides over 1000 Da in the hydrolysate had better abilities to donate electrons, whereas peptides below 1000 Da more effectively eliminated radicals. Therefore, peptides from freshwater clam extract could be employed as potential natural antioxidants, performing their activities via different mechanisms. Key words: Antioxidant peptides, freshwater clam extract, enzymatic hydrolysate, molecular weight distribution.

Evaluation Of Antioxidant Properties In Vitro of Plastein-Reaction-Stressed Soybean Protein Hydrolysate

Alcalase was used in the present study to carry out an enzymatic hydrolysis of soybean protein isolate and a plastein reaction of the prepared hydrolysate in vitro, aiming to investigate the influence of the plastein reaction on the antioxidant properties of the modified hydrolysate. Soybean protein hydrolysate was prepared in a degree of hydrolysis of 14.0%, exhibited a scavenging activity of 43.6% on ABTS radical in vitro, and thus was used as the substrate of the plastein reaction to prepare the plastein-reaction-stressed hydrolysate. Response surface methodology was applied to select suitable reaction conditions as follows: enzyme addition level 1037 U/g peptides, substrate concentration 29.7% (w/v), reaction temperature 20.3°C. The stressed hydrolysate showed the highest scavenging activity on ABTS radical (about 47.9%) or maximal reaction extent when reaction time was 6 h. Three stressed hydrolysates with different reaction extents were prepared and evaluated for other antioxidant activities. Compared to the original hydrolysate, the stressed hydrolysate with lower reaction extent exhibited a similar (P > 0.05) scavenging activity on DPPH (or superoxide) radical and reducing power, but a significant higher activity (P < 0.05) on hydroxyl radical. The stressed hydrolysate with the highest reaction extent behaved as these investigated antioxidant properties were significantly higher (P < 0.05) than the original hydrolysate except for scavenging activity on DPPH radical. The results of the present study highlight that the alcalase-catalyzed plastein reaction appears to be capable of improving antioxidant properties of soybean protein hydrolysate.

Glycation and transglutaminase mediated glycosylation of fish gelatin peptides with glucosamine enhance bioactivity

A mixture of novel glycopeptides from glycosylation between cold water fish skin gelatin hydrolysates and glucosamine (GlcN) via transglutaminase (TGase), as well as glycation between fish gelatin hydrolysate and GlcN were identified by their pattern of molecular distribution using MALDI-TOF-MS. Glycated/glycosylated hydrolysates showed superior bioactivity to their original hydrolysates. Alcalase-derived fish skin gelatin hydrolysate glycosylated with GlcN in the presence of TGase at 25°C (FAT25) possessed antioxidant activity when tested in a linoleic acid oxidation system, when measured according to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and when tested at the cellular level with human hepatocarcinoma (HepG2) cells as target cells. In addition, Alcalase-derived glycosylated hydrolysates showed specificity toward the inhibition of Escherichia coli (E. coli). The Flavourzyme-derived glycopeptides prepared at 37°C (FFC37 and FFT37) showed better DPPH scavenging activity than their native hydrolysates. The glycated Flavourzyme-derived hydrolysates were found to act as potential antimicrobial agents when incubated with E. coli and Bacillus subtilis.

Iron-binding properties of sugar cane yeast peptides

The extract of sugar-cane yeast (Saccharomyces cerevisiae) was enzymatically hydrolysed by Alcalase, Protex or Viscozyme. Hydrolysates were fractionated using a membrane ultrafiltration system and peptides smaller than 5kDa were evaluated for iron chelating ability through measurements of iron solubility, binding capacity and dialyzability. Iron-chelating peptides were isolated using immobilized metal affinity chromatography (IMAC). They showed higher content of His, Lys, and Arg than the original hydrolysates. In spite of poor iron solubility, hydrolysates of Viscozyme provided higher iron dialyzability than those of other enzymes. This means that more chelates of iron or complexes were formed and these kept the iron stable during simulated gastro-intestinal digestion in vitro, improving its dialyzability.

Ace Inhibition and Enzymatic Resistance In Vitro of a Casein Hydrolysate Subjected to Plastein Reaction in the Presence of Extrinsic Proline and Ethanol- or Methanol-Water Fractionation

Casein was hydrolyzed by alcalase to a degree of hydrolysis of 10.9% to obtain a hydrolysate having ACE-inhibition in vitro with an IC50 value of 52.6 μg/mL. The prepared hydrolysate was modified by alcalase-catalyzed plastein reaction with extrinsic proline added at 0.4 mol/mol free amino groups (on the basis of the hydrolysate), and fractionated by ethanol- or methanol-water solvents in proportions of 3:7, 5:5, or 7:3 (v/v), respectively. With the decrease of free amino groups of the modified hydrolysate as the response, the optimized plastein reaction conditions were alcalase addition of 3.1 kU/g peptides, substrate concentration of 50% (w/v), and reaction temperature of 25°C. Four modified hydrolysates prepared with different reaction times exhibited higher ACE-inhibitory activities than the original hydrolysate. The evaluation results showed that solvent fractionation of the modified hydrolysate with the maximum activity (IC50 = 13.0 μg/mL) yielded the separated soluble fraction's higher activity but the precipitate fraction's lower one. Further enzymatic digestion of the modified hydrolysate with the maximum activity and its two fractionated products by four proteases in vitro caused damage to the activities, but the residual activities of the final digests were higher than that of the original hydrolysate, indicating that the plastein reaction could confer casein hydrolysate protease resistance.

Characterization of oxalic acid pretreatment on lignocellulosic biomass using oxalic acid recovered by electrodialysis

The properties of pretreated biomass and hydrolysate obtained by oxalic acid pretreatment using oxalic acid recovered through electrodialysis (ED) were investigated. Most of the oxalic acid was recovered and some of the fermentation inhibitors were removed by ED. For the original hydrolysate, the ethanol production was very low and fermentable sugars were not completely consumed by Pichia stipitis during fermentation. Ethanol yield was less than 0.12g/g in all stage. For the ED-treated hydrolysate, ethanol production was increased by up to two times in all stages compared to the original hydrolysate. The highest ethanol production was 19.38g/l after 72h which correspond to the ethanol yield of 0.33g/g. Enzymatic conversion of the cellulose to glucose for all the pretreated biomass was in the range of 76.03 and 77.63%. The hydrolysis rate on each pretreated biomass was not significantly changed when oxalic acid recovered by ED was used for pretreatment.

Iron-chelating activity of chickpea protein hydrolysate peptides.

Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption.

Angiotensin I converting enzyme inhibition and enzymatic resistance in vitro

A casein hydrolysate was modified by the plastein reaction, fractionated with ethanol/water or methanol/water solvents and further hydrolyzed by four proteases to show the potential impact of these treatments on Angiotensin I converting enzyme (ACE) inhibition in vitro. The treated hydrolysate exhibited a higher ACE-inhibitory activity than did the original hydrolysate: the IC50 value decreased from 52.6 μg mL−1 (original) to 14.9 μg mL−1 (treated). Fractionation of the treated hydrolysate with the lowest polarity solvent led to soluble fractions with a higher activity and precipitate fractions with a lower activity. Hydrolysis of the treated or the fractionated hydrolysate by one of four selected proteases for 10 or 30 min reduced the activity; papain, pepsin or trypsin (but not alcalase) digests had residual activities of about 32–50% at 25 μg mL−1, higher than the original hydrolysate (27.8%). It was concluded that the plastein reaction, but not solvent fractionation, produced casein hydrolysates with protease resistance.

Evaluation of antioxidant activity of muscle and skin protein hydrolysates from giant kingfish, Caranx ignobilis (Forsskål, 1775)

SummaryTo assess the antioxidant properties of fish protein hydrolysate from giant kingfish Caranx ignobilis (Forsskål, 1775), muscle and skin were obtained using various proteases (papain, pepsin, trypsin and α‐chymotrypsin), respectively. Antioxidant activities of the resulting hydrolysates were evaluated using 2, 2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity, reducing power and metal chelating activity. Among the hydrolysates, peptic of muscle (37 ± 1.0; 0.290 ± 0.012; 63.30 ± 0.57) and tryptic of skin (36.2 ± 1.0; 0.190 ± 0.110; 63 ± 0.57) hydrolysates, which had the highest free radical scavenging activity, was subjected to purification using Sephadex G‐25. The fractionated hydrolysates were superior to the original hydrolysates in the antioxidative activity tested. The active fractions (MF2 and SF2) effectively inhibited lipid peroxidation in linoleic acid emulsion system, and the activity was compared with α‐tocopherol. The amino acid profile of MF2 and SF2 showed a high level of essential amino acids, and the most abundant amino acids were in the order His (12.01% and 7.08%), Phe (7.05% and 6.18%) and Lys (6.76% and 4.74%). Conclusively, C. ignobilis muscle and skin hydrolysates could be a promising rich source of natural antioxidants.

Sensory Characteristics and Antioxidant Activities of Maillard Reaction Products from Soy Protein Hydrolysates with Different Molecular Weight Distribution

Soy protein was hydrolyzed using two enzymes to obtain soy protein hydrolysate (SPH), the SPH was fractionated with ultrafiltration membranes to obtain peptide fractions below 1,000 Da (SP1) and 1,000–5,000 Da (SP2), and for the meantime, SPH was further completely hydrolyzed to get compound amino acids (CAA). Maillard reaction products (MRPs) were prepared from aqueous xylose–SPH/SP1/SP2/CAA model systems by heating at 120°C for 2.0 h. Compared with the original hydrolysates and other MRPs, the MRPs from SP2 exhibited a distinctly enhanced effect on flavor, including the caramel-like, soy sauce-like odors, umami and mouthful tastes and a greatly reduced bitterness in consomme´ soup. Antioxidant activities of SPH, SP1, SP2, CAA, and their MRPs were investigated through reducing power, DPPH radical-scavenging activity, and Fe2+ chelating activity. Before Maillard reaction, the antioxidant activities of peptide fractions with different molecular weights were quite different, and SP2 showed the highest activity; however, CAA exhibited very poor antioxidant activity. The antioxidant activities of SPH, SP1, SP2, and CAA were greatly enhanced by Maillard reaction, and the MRPs prepared from xylose–CAA model system exhibited a higher antioxidant activity than those from other model systems. Pyrazines, pyrroles, furans, and thiazoles were significantly correlated with reducing power and Fe2+ chelating activity by principal component analysis.

Biotechnological Production of Xylitol: Enhancement of Monosaccharide Production by Post-Hydrolysis of Dilute Acid Sugarcane Hydrolysate

Dilute-acid hydrolysis pretreatment of sugarcane bagasse resulted in release of 48% (18.4 g/L) of the xylan in the hemicellulose fraction into the hydrolysate as monomeric xylose. In order to enhance the recuperation of this monomer, a post-hydrolysis stage consisted of thermal treatment was carried out. This treatment resulted in an increase in xylose release of 62% (23.5 g/L) of the hemicellulose fraction. Original and post-hydrolysates were concentrated to the same levels of monomeric xylose in the fermentor feed. During the fermentation process, cellular growth was observed to be higher in the post-hydrolysate (3.5 g/L, Y(x/s) = 0.075 g cells/g xylose) than in the original hydrolysate (2.9 g/L, Y(x/s) = 0.068 g cells/g xylose). The post-treated hydrolysate required less concentration of sugars resulting in a lower concentration of fermentation inhibitors, which were formed primarily in the dilute acid hydrolysis step. Post-hydrolysis step led to a high xylose-xylitol conversion efficiency of 76% (0.7 g xylitol/g xylose) and volumetric productivity of 0.68 g xylitol/L h when compared to 71% (0.65 g xylitol/g xylose and productivity of 0.61 g xylitol/L h) for the original hemicellulosic hydrolysate.

Production of copper-chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin

Sunflower protein hydrolysates obtained with pepsin and pancreatin were used for purification of copper-chelating peptides by affinity chromatography with copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of β-carotene oxidation in the presence of copper. The protein hydrolysate obtained after 180 min incubation with pepsin plus 60 min with pancreatin was the most inhibitory of β-carotene oxidation. Purified chelating peptides were 2.5 times more antioxidant than the parent protein hydrolysate. Chelating peptides were enriched in certain amino acids, such as histidine and arginine, with respect to the original hydrolysate. This work shows that chelating peptides may be generated during digestion of sunflower proteins and have a protective role, due to their antioxidative activity, and favour mineral bioavailability.

Affinity Purification of Copper-Chelating Peptides from Sunflower Protein Hydrolysates

Copper-chelating peptides were purified from sunflower protein hydrolysates by affinity chromatography using immobilized copper. A variety of protein hydrolysates were obtained by incubation with the proteases Alcalase and Flavourzyme for different periods of time. Chelating activity was indirectly determined by measuring the inhibitory effect of hydrolysates on the oxidation of beta-carotene by copper. Copper-binding peptides purified from the two hydrolysates that inhibited oxidation by copper the most contained 25.4 and 42.0% histidine and inhibited beta-carotene oxidation 8 and 3 times more than the original hydrolysates, which had 2.4 and 2.6% histidine, respectively. Thus, histidine content is not the only factor involved in antioxidant activity, and probably other factors such as peptide size and amino acid sequence are also important. This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc. In addition to their antioxidant potential, chelating peptides are of nutritional interest because they increase bioavailability of minerals.