Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and Human Trypsins

Based on trypsin specificity (for lysines and arginines), trypsins from different sources are expected to hydrolyze a given protein to the same theoretical maximum degree of hydrolysis (DH_max,theo). This is in contrast with experiments. Using α-lactalbumin and β-casein, this study aims to reveal if the differences among experimental DH_max (DH_max,exp) by bovine, porcine, and human trypsins are due to their secondary specificity. Peptide analysis showed that ∼78% of all the cleavage sites were efficiently hydrolyzed by porcine trypsin, and ∼47 and ∼53% were efficiently hydrolyzed by bovine and human trypsins, respectively. These differences were explained by the enzyme secondary specificity, that is, their sensitivities to the amino acids around the cleavage sites. The DH_max predictions based on the secondary specificity were 4 times closer to the DH_max,exp than the predictions based on trypsin specificity alone (DH_max,theo). Proposed preliminary relations between binding sites and trypsin secondary specificity allow DH_max,exp estimations of tryptic hydrolysis of other proteins

Quantitative Fate of Chlorogenic Acid during Enzymatic Browning of Potato Juice

The quantitative fate of chlorogenic acid (ChA) during enzymatic browning of potato juice was investigated. Potato juice was prepared in water without the use of any antibrowning agent (<i>OX</i> treatment). As a control, a potato juice was prepared in the presence of NaHSO₃ (<i>S</i> control). To study the composition of phenolic compounds in potato in their native states, also a potato extract was made with 50% (v/v) methanol containing 0.5% (v/v) acetic acid (<i>MeOH</i> control). Water-soluble low molecular weight fractions (LMWFs) and high molecular weight fractions (HMWFs) from <i>S</i> and <i>OX</i> extracts were obtained by ultrafiltration and dialysis, respectively. Pellets obtained after the <i>OX</i> treatment and the <i>S</i> and <i>MeOH</i> controls were also analyzed for ChA content. Whereas in the <i>S-</i>LMWF all ChA was converted to sulfonic acid adducts, no free ChA was found in the <i>OX-</i>LMWF, indicating its high reactivity upon enzymatic browning. Analysis of protein in the HMWFs showed a higher content of “reacted” ChA in <i>OX</i> (49.8 ± 7.1 mg ChA/100 g potato DW) than in <i>S</i> (14.4 ± 1.5 mg ChA/100 g potato DW), as evidenced by quinic acid release upon alkaline hydrolysis. The presence of quinic acid in <i>S</i>-HMWF was unexpected, but a mass balance incorporating the ChA content of LMWF, HMWF, and pellet for the three extractions suggested that ChA might have been attached to polymeric material, soluble in the aqueous environment of <i>S</i> but not in that of <i>MeOH</i>. Size exclusion chromatography, combined with proteolysis, revealed that ChA reacted with patatin and protease inhibitors to produce brown soluble complexes

Effect of Soluble and Insoluble Fibers within the in Vitro Fermentation of Chicory Root Pulp by Human Gut Bacteria

The aim of this research was to study the in vitro fermentation of chicory root pulp (CRP) and ensiled CRP (ECRP) using human fecal inoculum. Analysis of carbohydrate levels in fermentation digests showed that 51% of all CRP carbohydrates were utilized after 24 h of fermentation. For ECRP, having the same cell wall polysaccharide composition as CRP, but with solubilization of 4 times more of CRP pectin due to ensiling, the fermentation was quicker than with CRP as 11% more carbohydrates were utilized within the first 12 h. The level of fiber utilization for ECRP after 24 h was increased by 8% compared to CRP. This effect on fiber utilization from ECRP seemed to arise from (i) increased levels of soluble pectin fibers (arabinan, homogalacturonan, and galactan) and (ii) ahypothesized more open structure of the remaining cell walls in ECRP, which was more accessible to degradation than the CRP cell wall network

Determination of the Influence of Substrate Concentration on Enzyme Selectivity Using Whey Protein Isolate and Bacillus licheniformis Protease

Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each cleavage site in the protein. It was determined from the identification and quantification of the peptides present in the hydrolysates. Solutions of 0.1–10% (w/v) whey protein isolate (WPI) were hydrolyzed by Bacillus licheniformis protease at constant enzyme-to-substrate ratio. The cleavage sites were divided into five groups, from very high (>10%) to very low selectivity (<0.1%). The selectivity toward cleavage sites after Glu 62 and 134 was 2 times higher at 10% (w/v) WPI than at the lower protein concentrations. This finding shows that both the rate of hydrolysis and the enzyme selectivity were influenced by the substrate concentration

Analysis of Palmitoyl Apo-astaxanthinals, Apo-astaxanthinones, and their Epoxides by UHPLC-PDA-ESI-MS

Food products enriched with fatty acid-esterified xanthophylls may result in deviating dietary apo-carotenoids. Therefore, free astaxanthin and its mono- and dipalmitate esters were subjected to two degradation processes in a methanolic model system: light-accelerated autoxidation and hypochlorous acid/hypochlorite (HOCl/OCl^–) bleaching. Reversed phase ultrahigh-performance liquid chromatography photodiode array with in-line electrospray ionization mass spectrometry (RP-UHPLC-PDA-ESI-MS) was used for assessment of degradation products. Apo-astaxanthinals and -astaxanthinones containing 3 (apo-9) to 10 (apo-8′) conjugated double bonds were found upon autoxidation for all three types of astaxanthin (except free apo-8′-astaxanthinal). Fragmentation of [M + H]⁺ and [M + Na]⁺ parent masses of apo-astaxanthins from dipalmitate astaxanthin indicated palmitate esterification. Astaxanthin monopalmitate degradation resulted in a mixture of free and palmitate apo-astaxanthins. HOCl/OCl^– rapidly converted the astaxanthins into a mixture of epoxy-apo-9- and epoxy-apo-13-astaxanthinones. The palmitate ester bond was hardly affected by autoxidation, whereas for HOCl/OCl^– the ester bond of the apo-astaxanthin palmitoyl esters was degraded

Separation and Identification of Individual Alginate Oligosaccharides in the Feces of Alginate-Fed Pigs

This research aimed to develop a method for analyzing specific alginate oligosaccharides (AOS) in a complex matrix such as pig feces. The data obtained were used to study alginate degradation by the microbiota in the large intestine during adaptation, including the individual variation between pigs. A method using an UHPLC system with an ethylene bridged hybrid (BEH) amide column coupled with MS^<i>n</i> detection was able to distinguish saturated and unsaturated AOS with DP 2–10. Isomers of unsaturated trimer and tetramer could be separated and annotated. In the feces, saturated and unsaturated AOS were present. The presence of unsaturated AOS indicates that the microbiota produced alginate lyase. The microbiota utilized unsaturated AOS more than saturated AOS. The results also suggested that guluronic acid at the reducing end of AOS inhibits the utilization by microbiota during the first weeks of adaptation. After adaptation, the microbiota was able to utilize a broader range of AOS

Efficacy of Food Proteins as Carriers for Flavonoids

Enrichment of flavonoids in food is often limited by their off-tastes, which might be counteracted by the use of food proteins as carriers of flavonoids. Various milk proteins, egg proteins, and gelatin hydrolysates were compared for their binding characteristics to two flavan-3-ols. Among the proteins tested for their affinities toward epigallocatechin gallate (EGCG), β-casein and gelatin hydrolysates, in particular fish gelatin, were found to be the most promising carriers with an affinity on the order of 10⁴ M^–1. A flexible open structure of proteins, as present in random coil proteins, was found to be important. The saturation of binding observed at high flavonoid/protein ratios was used to estimate the maximal binding capacity of each protein. To reach a daily intake of EGCG that has been associated with positive health effects, only 519 mg of gelatin B and 787 mg of β-casein were required to complex EGCG on the basis of their maximal binding capacity. When the absence of turbidity is taken into account, β-casein prevails as carrier. Three selected proteins were further investigated for their binding potential of representative flavonoids differing in their C-ring structure. An increase in hydrophobicity of flavonoids was related to a higher affinity for proteins, and the presence of a gallic acid ester on the C-ring showed an overall higher affinity

Py-GC/MS pyrograms of wheat straw, basic compost mix (BM) and compost after 16 days of mycelium growth (PIII–16) (A) and water un-extractable solids (WUS) of Phase I and PIII–16 (B) for batch A.

<p>The identities and structures of main syringyl and guaiacyl (and p-hydroxyphenyl) compounds are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138909#pone.0138909.g002" target="_blank">Fig 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138909#pone.0138909.s001" target="_blank">S1 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138909#pone.0138909.s002" target="_blank">S2 Table</a>. PI: compost after Phase I: PII: compost after Phase II.</p

Modification of Prenylated Stilbenoids in Peanut (<i>Arachis hypogaea</i>) Seedlings by the Same Fungi That Elicited Them: The Fungus Strikes Back

<i>Aspergillus oryzae</i> and <i>Rhizopus oryzae</i> were compared for inducing the production of prenylated stilbenoids in peanut seedlings. The fungus was applied at two different time points: directly after soaking (day 1) or after 2 days of germination (day 3). <i>Aspergillus</i>- and <i>Rhizopus</i>-elicited peanut seedlings accumulated an array of prenylated stilbenoids, with overlap in compounds induced, but also with compounds specific to the fungal treatment. The differences were confirmed to be due to modification of prenylated stilbenoids by the fungus itself. Each fungus appeared to deploy different strategies for modification. The content of prenylated stilbenoids modified by fungi accounted for around 8% to 49% (w/w) of total stilbenoids. The contents of modified prenylated stilbenoids were higher when the fungus was applied on day 1 instead of day 3. Altogether, type of fungus and time point of inoculation appeared to be crucial parameters for optimizing accumulation of prenylated stilbenoids in peanut seedlings

Mass Spectrometric Characterization of Benzoxazinoid Glycosides from Rhizopus-Elicited Wheat (Triticum aestivum) Seedlings

Benzoxazinoids function as defense compounds and have been suggested to possess health-promoting effects. In this work, the mass spectrometric behavior of benzoxazinoids from the classes benzoxazin-3-ones (with subclasses lactams, hydroxamic acids, and methyl derivatives) and benzoxazolinones was studied. Wheat seeds were germinated with simultaneous elicitation by Rhizopus. The seedling extract was screened for the presence of benzoxazinoid (glycosides) using reversed-phase ultra-high-performance liquid chromatography with photodiode array detection coupled in line to multiple-stage mass spectrometry (RP-UHPLC–PDA–MSⁿ). Benzoxazin-3-ones from the different subclasses showed distinctly different ionization and fragmentation behaviors. These features were incorporated into a newly proposed decision guideline to aid the classification of benzoxazinoids. Glycosides of the methyl derivative 2-hydroxy-4-methoxy-1,4-benzoxazin-3-one were tentatively identified for the first time in wheat. We conclude that wheat seedlings germinated with simultaneous fungal elicitation contain a diverse array of benzoxazinoids, mainly constituted by benzoxazin-3-one glycosides