Protein hydrolysates from Bluefin Tuna (Thunnus thynnus) heads as influenced by the extent of enzymatic hydrolysis

Functional properties and antioxidant activities of protein hydrolysates from tuna (Thunnus thynnus) heads (THPHs), with different degrees of hydrolysis, obtained by treatment with Bacillus mojavensis A21 alkaline proteases and Alcalase, were investigated. Protein content of all freeze-dried THPHs ranged from 73.74 ± 0.5 to 78.56 ± 1.2%. The THPHs had excellent solubility, compared to untreated tuna head proteins and possessed interfacial properties, which were governed by their concentrations. Similarly, at a degree of hydrolysis (DH) of 12 and 15%, > 90% nitrogen solubility was observed at all experimental pH values tested. The emulsifying activity index (EAI) and emulsion stability index (ESI) of both hydrolysates at different DHs decreased (p < 0.05) with increasing DH. At low DH (5%), hydrolysates exhibited strong emulsifying properties. All THPHs produced by the A21 proteases generally showed higher antioxidative activity than that of the Alcalase protein hydrolysates. The highest DPPH radical-scavenging activity (78 ± 2.1% at 3 mg/mL) was obtained with a DH of 15%. The IC₅₀ value for the β-carotene bleaching assay was 0.5 ± 0.03 mg/mL. Alcalase (DH = 12%) and A21 (DH = 15%) protein hydrolysates contained glutamic acid/glutamine and arginine as the major amino acids, followed by lysine, aspartic acid/ asparagine, histidine, valine, phenylalanine, and leucine. In addition, the THPHs had a high percentage of essential amino acids, which made up 50.52 and 50.47%, of the protein hydrolysates obtained by the Alcalase and A21 proteases, respectively. Therefore, THPHs can be used as a promising source of functional peptides with antioxidant properties.     

Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates

Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides. The antioxidant activities of the hydrolysates were measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. Among hydrolysates, pepsin hydrolysate obtained by 3 h exhibited the highest antioxidant activities, which could also quench the hydroxyl radical, chelate ferrous ion, exhibit reducing power and inhibit the lipid peroxidation. Then, 3-h pepsin hydrolysates were purified sequentially by ultrafiltration, gel filtration and RP-HPLC. The sequence of the peptide with the highest antioxidative activity was identified to be Ala-Asp-Ala-Phe (423.23 Da) using RP-HPLC-ESI-MS, which was identified for the first time from walnut protein hydrolysates. Last, the inhibition of the peptide on lipid peroxidation was similar with that of reduced glutathione (GSH). These results indicate that the protein hydrolysates and/or its isolated peptides may be effectively used as food additives.     

Structural characteristics of hydrolysates of proteins from extracted sunflower flour at the air-water interface

The structural and topographical characteristics of a sunflower protein isolate (SPI) and its hydrolysates at different degrees of hydrolysis (DH = 5.62%, 23.5%, and 46.3%) spread at the air-water interface at pH 7 and 20 degrees C were determined from pi-A isotherms coupled with Brewster angle microscopy (BAM). The structural characteristics of SP hydrolysate spread monolayers depend on the degree of hydrolysis. We observed a significant shift of the pi-A(APPARENT) isotherms toward lower molecular areas as the degree of hydrolysis (DH) increased. This phenomenon was attributed to spreading of the protein at the interface, especially at DH 46.3%. A change in the monolayer structure was observed at a surface pressure of 12-15 mN/m. At a microscopic level, the heterogeneous monolayer structures visualized near the monolayer collapse and during the monolayer expansion proved the existence of large regions of protein aggregates. Reflectivity increased with surface pressure and was a maximum at the monolayer collapse. The monolayer thickness decreased as the degree of hydrolysis increased. These phenomena explain the poor functional properties for the formation and stabilization of a dispersion (emulsion or foam) of protein hydrolysates at high degrees of hydrolysis.     

Peptides and hydrolysates from casein and soy protein modulate the release of vasoactive substances from human aortic endothelial cells

Food proteins were shown to affect atherogenic risk factors, which is supposed to be related to specific peptide sequences encrypted within their primary sequence. The aim of this study was to evaluate the effects of peptides and hydrolysates from two food proteins, casein and soy protein, on endothelial cell functions (cell proliferation and release of vasoactive substances). Cell proliferation was not influenced by dipeptides and most of the tripeptides, whereas several total hydrolysates from casein and soy protein inhibited cell proliferation at higher concentrations (>0.25 mg/mL; P<0.05). The release of one or more of the vasoactive substances, thromboxan B2 (stable marker of thromboxan A2), 6-keto-prostaglandin F1alpha (stable marker of prostaglandin I2), endothelin-1, and nitric oxide, was significantly influenced by the incubation with various peptides compared with control cells (P<0.05). Various hydrolysate fractions from casein and soy protein influenced the release of 6-keto-prostaglandin F1alpha and nitric oxide (P<0.05) but did not influence the release of thromboxan B2 and endothelin-1. In conclusion, the present study demonstrates that peptides and hydrolysate fractions from casein and soy protein influence endothelial cell function as evidenced by the modulation of endothelial cell proliferation and alterations in the release of vasoactive substances.     

Biotransformation of fatty acid-rich tree oil hydrolysates to hydroxy fatty acid-rich hydrolysates by hydroxylases and their feasibility as biosurfactants

The fatty acid compositions of 10 types of tree oils were analyzed and Camellia japonica (CJ), Tetradium daniellii (TD), and Hovenia dulcis (HD) tree oils were selected to be oleic acid (OA)-, linoleic acid (LA)-, and α-linoleic acid (ALA)-rich tree oils, respectively. Recombinant Escherichia coli expressing 10-hydratase and 7,8-diol synthase converted 31.7 and 15.6 g/L unsaturated fatty acids (UFAs) in OA-rich oil hydrolysates to 21.7 g/L 10-monohydroxy fatty acid (monoHFA) and 13.3 g/L 7,8-diHFA, respectively. The cells expressing 13-hydratase, 13-lipoxygenase, 5,8-diol synthase, and 8,11-diol synthase converted 42.8, 28.5, 10.0, and 20.0 g/L UFAs in LA-rich oil hydrolysates to 28.2 g/L 13-monoHFA, 11.8 g/L 13-monoHFA, 7.2 g/L 5,8-diHFA, and 8.9 g/L 8,11-diHFA, respectively. The cells expressing 8,11-diol synthase converted containing 17.5 g/L UFAs in ALA-rich oil hydrolysate to 7.5 g/L 8,11-diHFA. The average emulsifying activities of diHFArich and monoHFA-rich tree oil hydrolysates were 13.9- and 4.3-fold higher than those of tree oil hydrolysates, respectively. Thus, HFA-rich tree oil hydrolysates derived from tree oils can be applied as biosurfactants, and the fatty acid-rich residue as by-product obtained from the tree refinery process may be recycled into biosurfactants.     

Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

The bioactivity and fractionation of peptide hydrolysates in cultures of CHO cells

Peptide hydrolysate supplements in mammalian cell cultures provide enhanced growth and productivity. The objective of this study was to compare the bioactivity of ten different commercially available hydrolysates from plant, microbial, and animal sources. The peptide hydrolysates were tested as supplements to cultures of Chinese hamster ovary (CHO) cells that produce human beta interferon (β‐IFN). A soy hydrolysate was shown to support high cell growth but not protein productivity compared to an animal component hydrolysate (Primatone RL). On the other hand, a yeast hydrolysate showed lower cell growth, but comparable productivity of the recombinant protein. Glycosylation analysis showed that the glycan profile of β‐IFN produced in yeast hydrolysate supplemented cultures was equivalent to that from Primatone RL‐supplemented cultures. Fractionation of the yeast hydrolysate and Primatone RL produced a similar protein‐assayed pattern except for one extra peak at around 1 kDa in the Primatone RL profile. A fraction taken at a molecular weight range of 1.5–1.7 kDa showed the highest growth promoting activity in both samples. However, four other fractions in yeast hydrolysate and two in Primatone RL at lower molecular weights showed some growth promoting activity. In conclusion, the yeast hydrolysates provided a good alternative to the animal sourced Primatone RL for high productivity of β‐IFN from CHO cells with equivalent glycosylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:584–593, 2014     

Detoxification of hemicellulosic hydrolysates from extracted olive pomace by diananofiltration

Xylitol can be obtained from the pentose-rich hemicellulosic fraction of agricultural residues, such as extracted olive pomace, by fermentation. Dilute acid hydrolysis of lignocellulosic materials, produces the release of potential inhibitory compounds mainly furan derivatives, aliphatic acids, and phenolic compounds. In order to study the potential on the increase of the hydrolysate fermentability, detoxification experiments based on diananofiltration membrane separation processes were made. Two membranes, NF270 and NF90, were firstly evaluated using hydrolysate model solutions under total recirculation mode, to identify the best membrane for the detoxification. NF270 was chosen to be used in the diananofiltration experiment as it showed the lowest rejection for toxic compounds and highest permeate flux. Diananofiltration experiments, for hydrolysate model solutions and hydrolysate liquor, showed that nanofiltration is able to deplete inhibitory compounds and to obtain solutions with higher xylose content. Conversely to non-detoxified hydrolysates, nanofiltration detoxified hydrolysates enabled yeast growth and xylitol production by the yeast Debaryomyces hansenii, clearly pointing out that detoxification is an absolute requirement for extracted olive pomace dilute acid hydrolysate bioconversion.     

Sequence Determination of an Antioxidant Peptide Obtained by Enzymatic Hydrolysis of Oyster <Emphasis Type="Italic">Crassostrea madrasensis</Emphasis> (Preston)

Soft tissue from cultured farm fresh oysters (Crassostrea madrasensis) was subjected to two standard enzymatic peptide extraction procedures using pepsin and papain. The crude extracts obtained were partially purified by column chromatography and were freeze-dried. The hydrolysates obtained were compared with respect to their degree of hydrolysis (DH), antioxidant potential (AP) and total phenolic content (TPC). The hydrolysate showing better antioxidant property was further subjected to purification by high performance liquid chromatography and characterized by LC-MS/MS. Papain-digested oyster protein (OPHpap) hydrolysate showed higher DH, AP and TPC. OPHpap was further subjected to ultrafiltration and fractionated into 3 sizes namely, above 10, 3–10 and 1–3 kDa according to the molecular size. Antioxidant capacity of <3 kDa fraction OPHpap-3 evaluated by DPPH free radical scavenging assay, metal chelating activity, linoleic acid autoxidation assay showed maximum effectiveness. Of the seven fractions collected by purification of OPH-pap-3 on semi-preparative RP-HPLC, fraction 7 that showed the highest antioxidant activity was further characterized by LC-ESI-MS/MS and its sequence determined. An antioxidant peptide molecule with thirteen amino acids was identified in oyster protein hydrolysate obtained by papain digestion that may find application as a nutraceutical or may be utilized in food industry for prevention of rancidity in foods.     

Biological detoxification of different hemicellulosic hydrolysates using Issatchenkia occidentalis CCTCC M 206097 yeast

This work had as its main objective to contribute to the development of a biological detoxification of hemicellulose hydrolysates obtained from different biomass plants using Issatchenkia occidentalis CCTCC M 206097 yeast. Tests with hemicellulosic hydrolysate of sugarcane bagasse in different concentrations were carried out to evaluate the influence of the hydrolysate concentration on the inhibitory compounds removal from the sugarcane bagasse hydrolysate, without reduction of sugar concentration. The highest reduction values of inhibitors concentration and less sugar losses were observed when the fivefold concentrated hydrolysate was treated by the evaluated yeast. In these experiments it was found that the high sugar concentrations favored lower sugar consumption by the yeast. The highest concentration reduction of syringaldehyde (66.67%), ferulic acid (73.33%), furfural (62%), and 5-HMF (85%) was observed when the concentrated hydrolysate was detoxified by using this yeast strain after 24 h of experimentation. The results obtained in this work showed the potential of the yeast Issatchenkia occidentalis CCTCC M 206097 as detoxification agent of hemicellulosic hydrolysate of different biomass plants.     

Peptide and amino acid composition of different protein bases for culture media

The enzymatic hydrolysates under study, obtained from different raw materials, have been shown to contain a great variety of peptides with different molecular weight. The highest content of fractions with a molecular weight of 2000 D has been observed in enzymatic meat and casein hydrolysates manufactures in the GDR. Low-molecular fractions (100-200 D) prevail in amino peptide. A great variety of peptides with different molecular weight is observed in Hottinger's meat hydrolysate and in blood clot hydrolysate obtained from the blood of laboratory animals. All peptide fractions have been shown to contain a wide spectrum of free amino acids. These data on the peptide and amino acid composition of different protein bases facilitate their rational use of microbiological culture media.     

Enzymatic hydrolysis of whey proteins. II. Molecular-weight range

Using high-pressure liquid chromatography we studied the distribution of molecular weights in whey-protein hydrolysates using the following commercially obtained proteases: Alcalasa 0.6 L and Protease 660 L, both bacterial in origin, and PEM 2500 S, of animal origin. In each of the systems, the range of molecular weights in the hydrolysate depended solely on the degree of hydrolysis (DH) achieved. For DH >/= 20, between 65% and 95% of the hydrolysate is made up of peptides with a molecular weight of less than 1,000 Da. (c) 1994 John Wiley & Sons, Inc.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture

To investigate the effect of size-excluded fraction of non-animal protein hydrolysate on growth, viability and longevity of Chinese hamster ovary (CHO) cells, several commercially available protein hydrolysates were evaluated as a feed supplement to chemically-defined protein-free suspension culture. Soy protein hydrolysates showed better supporting capability for cell growth and viability than the other types of hydrolysates. Maximal cell growth was not affected greatly by size exclusion of some soy hydrolysates such as bacto soytone and soy hydrolysates. CHO cells supplemented with size-excluded fractions of the two hydrolysates showed viable cell density and viability almost equal to those with their crude hydrolysates, although soy hydrolysates showed a little better performance. This suggested that the size-excluded hydrolysate fractions of some soy hydrolysate might be a potential culture medium additive to achieve better downstream operation in a large-scale production as well as enhanced productivity.     

Antioxidant peptides isolated from the marine rotifer,Brachionus rotundiformis

Protein derived from the rotifer Brachionus rotundiformis was hydrolyzed using different proteases (Alcalase, α-chymotrypsin, Neutrase, papain, pepsin and trypsin) for production of antioxidant peptide. Antioxidant activities of hydrolysates were evaluated using DPPH radical scavenging activity. Peptic hydrolysate exhibited the highest antioxidative activity compared to other hydrolysates. To identify antioxidant peptides, peptic hydrolysate was purified using consecutive chromatographic methods, and antioxidant peptides were identified to be Leu-Leu-Gly-Pro-Gly-Leu-Thr-Asn-His-Ala (1076 Da), and Asp-Leu-Gly-Leu-Gly-Leu-Pro-Gly-Ala-His (1033 Da) by Q-TOF ESI mass spectroscopy. EC₅₀ values of purified peptides were 189.8 and 167.7 μM, respectively. Antioxidant activities of peptides purified from the rotifer protein hydrolysate were evaluated, with results showing that peptides significantly quenched free radicals.     

Chemical composition and theoretical nutritional evaluation of the produced fractions from enzymic hydrolysis of salmon frames with Protamex™

An amount of 200 kg fresh salmon frames were enzymic hydrolysed with the commercial protease mixture Protamex™, which is known to produce non-bitter hydrolysates. After the enzymic procedure the frames were separated by centrifugation into five fractions: an aqueous fraction rich in peptides, an insoluble fraction, an emulsion fraction, salmon oil and a bone fraction. Approximately 48% of total crude protein present in the salmon frames were found in the aqueous fraction, in which the lipid content was reduced to <0.1% in dry samples after ultramembrane filtration (UF fraction). The UF fraction was low in tryptophan, leucine and phenylalanine+tyrosine, but high in taurine. Nearly 19% of total crude protein present in the salmon frames were found in the insoluble fraction. This fraction was high in most of the indispensable amino acids. Approximately 77% of total lipids present in the salmon frames were isolated as salmon oil, which was high in both eicosapentaenic acid (EPA) and docosahexaenic acid (DHA). The bone fraction contained 62% of total ash present in the salmon frames and was high in the minerals Ca, P and Mg and also in the trace elements Cu, Fe, I, Mn, Se and Zn. All of the produced fractions were low in the undesirable substances As, Cd, Hg and Pb. For future studies the UF fraction and salmon oil might be interesting as health promoting agents, the insoluble fraction as dietary protein supplement and the bone fraction as dietary mineral supplement.     

Dipeptidyl peptidase IV inhibitory and antioxidative properties of milk protein-derived dipeptides and hydrolysates

Selected synthetic dipeptides and milk protein hydrolysates were evaluated for their dipeptidyl peptidase IV (DPP-IV) inhibitory properties, and their superoxide (SO) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities. DPP-IV inhibition was seen with eight out of the twelve dipeptides and 5 of the twelve hydrolysates studied. Trp-Val inhibited DPP-IV, however, inhibition was not observed with the reverse peptide Val-Trp. The most potent hydrolysate inhibitors were generated from casein (CasH2) and lactoferrin (LFH1). Two Trp containing dipeptides, Trp-Val and Val-Trp, and three lactoferrin hydrolysates scavenged DPPH. The dipeptides had higher SO EC₅₀ values compared to the milk protein hydrolysates (arising from three lactoferrin and one whey protein hydrolysates). Higher molecular mass fractions of the milk protein hydrolysates were associated with the SO scavenging activity. Trp-Val and one lactoferrin hydrolysate (LFH1) were multifunctional displaying both DPP-IV inhibitory and antioxidant (SO and DPPH scavenging) activities. These compounds may have potential as dietary ingredients in the management of type 2 diabetes by virtue of their ability to scavenge reactive oxygen species and to extend the half-life of incretin molecules.     

Sugar recovery and fermentability of hemicellulose hydrolysates from steam-exploded softwoods containing bark

The hemicellulose sugar recovery and ethanol production obtained from SO2-catalyzed steam explosion of a mixed white fir (70%) and ponderosa pine (30%) feedstock containing bark (9% dry weight/dry weight) was assessed. More than 90% of the available hemicellulose sugars could be recovered in the hydrolysate obtained after steam explosion at 195 degrees C, 2.38 min, and 3.91% SO2, with 59% of the original hemicellulose sugars detected in a monomeric form. Despite this high sugar recovery, this hydrolysate showed low ethanol yield (64% of theoretical yield) when fermented with a spent sulfite liquor-adapted strain of Saccharomyces cerevisiae. In contrast, most hydrolysates prepared at higher steam explosion severity showed comparable or higher ethanol yields. Furthermore, the hydrolysates prepared from bark-free feedstock showed better fermentability (87% of theoretical yield) despite containing higher concentration of known inhibitors. The ethanol yield from the hydrolysate prepared from a bark-containing wood sample could be improved to 81% by an extra stage acid hydrolysis (121 degrees C for 1 h in 3% sulfuric acid). This extra stage acid hydrolysis and steam explosion at higher severity conditions seem to improve the fermentability of the hydrolysates by transforming certain inhibitory compounds present in the hydrolysates prepared from the bark-containing feedstock and thus lowering their inhibitory effect on the yeast used for the ethanol fermentation.     

Hydrolysis of rapeseed protein isolates: Kinetics,characterization and functional properties of hydrolysates

Two rapeseed protein isolates corresponding to albumins and globulins, respectively, were produced from an industrial defatted rapeseed meal. A pilot-scale process of protein extraction has been developed to remove major anti-nutritional compounds, easy to scale-up and using recyclable solvents. The kinetics of the hydrolyses of these two protein substrates using Alcalase 2.4L^® were compared by the measurement of the degree of hydrolysis (DH) when varying the initial proteins and enzyme concentrations. The globulins isolate was hydrolysed more efficiently than the albumins isolate mainly due to the compact and globular conformation of albumin (napin). Kinetic parameters have been determined for both substrates and a log-linear relation has been established between the DH values at a definite time and the initial enzyme/substrate ratio. Such relationships allow an effective monitoring of hydrolysis process since the hydrolysates analysis using reverse-phase chromatography coupled with mass spectrometry revealed that peptide maps corresponding to peptides of molecular weight inferior to 1 kDa are similar at a specific DH, independently of the reaction temperature and initial concentrations of substrate and enzyme. Thus, it is demonstrated that the DH is the sole parameter needed to control the physico-chemical properties and consequently the functionalities (solubility, foaming and emulsifying properties) which depend on the nature of peptides present in the hydrolysates.