In vitro digestibility and solubility of phosphorus of three plant-based meat analogues.

Interest in plant-based meat analogues has increased and can be expected to be applied to pet foods, which necessitates the understanding of the nutrient supply in those foods. Our primary aim was to advance our understanding of the digestive properties of sterilized plant-based meat analogues. The impact of the preparatory processing steps on the solubility of meat analogues was studied. Meat analogues were made by mixing water, salt, and wheat gluten with soy protein isolate, pea protein isolate, or faba bean concentrate. Mixed materials were processed into model meat analogues using shear cell technology. Products were canned in water or gravy and sterilized. An animal-based canned pet food was made as a reference. Products sampled at the processing steps (mixing, shearing, sterilization) were digested in vitro. Samples of digestate were taken at the gastric phase (0 and 120 min) and small intestinal phase (120, 200, 280, and 360 min) for analysis of protein hydrolysis. The extent digestion of nitrogen and dry matter was determined at the end of incubation. Total phosphorus, soluble phosphorus after acid treatment, and after acid and enzymatic treatment were determined. The degree of hydrolysis after gastric digestion was low but increased immediately in the small intestinal phase; products based on pea had the highest values (56%). Nitrogen digestibility was above 90% for all materials at each processing step, indicating that bioactive compounds were absent or inactivated in the protein isolates and concentrate. Phytate seemed to play a minor role in meat analogues, but phosphorus solubility was influenced by processing. Shearing decreased soluble phosphorus, but this effect was partly reversed by sterilization. Nutrient digestibility as well as phosphorus solubility in plant-based products was higher than or comparable with the reference pet food. These findings show that the digestive properties of the tested plant-based meat analogues do not limit the supply of amino acids and phosphorus.

UVB pretreatment of ß-lactoglobulin affects the temperature-induced formation of functional amyloid-like aggregates and promotes oxidative degradation.

Unfolding in combination with or without acid hydrolysis is crucial for the formation of functional amyloid (fibrillar) or amyloid-like (worm-like) ß-lactoglobulin (BLG) aggregates, which can be induced through temperature treatment for several hours at pH 2-4. A preceding conformational destabilization of BLG might affect its aggregation. We investigated ultraviolet (UV) B radiation as conformational perturbing treatment to facilitate temperature-induced protein aggregation. 2-h UVB pretreated BLG (UV-BLG) exhibited an accelerated worm-like aggregation at pH 3.5, while at pH 2 the formation of fibrils was decelerated. The UV-induced conformational destabilization lowered the thermal stability and thus facilitates unfolding during thermal treatment. Thereby, the formation of covalent and non-covalent intermolecular interactions was favored, which promoted assembly of intact proteins resulting in worm-like aggregates. The oxidative degradation of UV-BLG was suggested to alter fibrillation-prone protein regions and thereby impede peptide assembly.

Modification and oxidative degradation of ß-lactoglobulin by UVB irradiation.

Ultraviolet (UV) B irradiation induces protein modification, especially the conformational rearrangement of proteins, and is therefore promising as a non-thermal and non-chemical functionalization technique. Nevertheless, UVB irradiation introduces radicals and oxidizes side chains resulting in the loss of food quality. Thus, assessing the UVB irradiation-based functionalization of ß-lactoglobulin (BLG) versus its oxidative degradation is of interest. UVB irradiation of up to 8 h was successfully applied to loosen the rigid folding of BLG and increase its flexibility. Thereby, the cysteine at position 121 and hydrophobic regions became surface-exposed as indicated by the increase in accessible thiol groups and increased surface hydrophobicity. Furthermore, we demonstrated the cleavage of the "outer" disulfide bond C66-C160 by LC-MS/MS after tryptic digestion of BLG. The 2-h-irradiated BLG showed adequate conformational rearrangement for protein functionalization while being minimally oxidized.

A novel screening method for free non-standard amino acids in human plasma samples using AccQ·Tag reagents and LC-MS/MS.

There are at least 500 naturally occurring amino acids, of which only 20 standard proteinogenic amino acids are used universally across all organisms in the synthesis of peptides and proteins. Non-standard amino acids can be incorporated into proteins or are intermediates and products of metabolic pathways. While the analysis of standard amino acids is well-defined, the analysis of non-standard amino acids can be challenging due to the wide range of physicochemical properties, and the lack of both reference standards and information in curated databases to aid compound identification. It has been shown that the use of an AccQ·Tag™ derivatization kit along with LC-MS/MS is an attractive option for the analysis of free standard amino acids in complex samples because it is fast, sensitive, reproducible, and selective. It has been demonstrated that the most abundant quantitative transition for MS/MS analysis of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids corresponds to the fragmentation of the molecule at the 6-aminoquinoline carbonyl group producing a common m/z 171 fragment ion and occurs at similar mass spectrometry collision energy and cone voltages. In this study, the unique properties of AQC derivatized amino acids producing high intensity common fragment ions, along with chromatographic separation of amino acids under generic chromatography conditions, were used to develop a novel screening method for the detection of trace levels of non-standard amino acids in complex matrices. Structural elucidation was carried out by comparing the MS/MS fragment ion mass spectra generated with in silico predicted fragmentation spectra to enable a putative identification, which was confirmed using an appropriate analytical standard. This workflow was applied to screen human plasma samples for bioactive thiol-group modified cysteine amino acids and S-allylmercaptocysteine (SAMC), S-allylcysteine sulfoxide (SACS or alliin) and S-propenylcysteine (S1PC) are reported for the first time to be present in human plasma samples after the administration of garlic supplements.

Adjustment of triple shellac coating for precise release of bioactive substances with different physico-chemical properties in the ileocolonic region.

Formulations for the controlled release of substances in the human terminal ileum and colon are essential to target the gut microbiome and its interactions with the intestinal mucosa. In contrast to pharmaceutical enteric coatings, reliable food-grade alternatives are still scarce. Shellac coatings have been used for various active ingredients, but their stability is affected by the physicochemical properties of the encapsulated substances. It is well known, that shellac release can be modulated by an acidic subcoating. Here, we hypothesized that a triple shellac coating with an adjusted intermediate coating (acidic or alkaline) can be effectively used to counteract the differences in pH value of various encapsulated substances, allowing a precise targeting of the desired release pH value. First, the system was tested with riboflavin 5'-monophosphate sodium salt dihydrate (RMSD) as a characteristic model substance. Secondly, it was transferred to nicotinic acid (NA) and nicotinamide (NAM) as bioactive compounds with different physio-chemical properties: NAM, an alkaline crystalline and highly water-soluble substance, led to a premature release from conventional shellac microcapsules, whereas RMSD and NA with their medium solubility and neutral to acidic pH properties delayed the shellac dissolution. A precise modulation of the release profile of each substance was possible by the addition of different intermediate subcoatings: an acidic layer with citric acid counteracted the premature release of the alkaline and highly soluble NAM. In contrast, an alkaline sodium bicarbonate intermediate subcoating enhanced shellac swelling and delayed the release of NA and RMSD. In conclusion, the novel triple-layer shellac coating provides a much higher adaptability and reliability for nutritional formulations aiming at a targeted release in the ileocolonic region.

ß-Lactoglobulin as nanotransporter for allicin: Sensory properties and applicability in food.

The thiosulfinate allicin is a labile, bioactive compound of garlic. In order to enrich allicin in a functional food, a delivery system which stabilises the compound and masks its intense flavour is necessary. In the present study allicin was covalently bound to the whey protein ß-lactoglobulin and the incorporation of this transporter in a food matrix was tested. The sensory properties of the pure functional ingredient as well as of an enriched beverage were characterised by quantitative descriptive analysis. The concentration of volatile compounds was analysed by headspace gas chromatography-mass spectrometry. The garlic-related organoleptic properties of garlic powder were significantly improved by the binding of allicin in combination with spray drying. After purification of the modified ß-lactoglobulin the garlic taste and smell were barely perceptible. ß-Lactoglobulin modified with allicin provided a stable functional ingredient that can be used to enrich a broad range of food products.

ß-Lactoglobulin as nanotransporter--Part I: Binding of organosulfur compounds.

The binding reaction of allicin and diallyl disulfide with ß-lactoglobulin and the influence of pH value and protein denaturation on this reaction have been examined in the present study. Regardless of the structural similarity of both the organosulfur compounds, their binding behavior was significantly different. Both ligands were covalently bound by the free thiol group of the protein, whereas the affinity for allicin was significantly higher. In addition, diallyl disulfide was non-covalently bound. The binding reaction of both ligands was very sensitive to the pH value during incubation. The optimal pH range was between pH 8.0 and 9.0. Protein denaturation increased the reaction rate and reduced the number of binding sites for allicin, whereas the number of non-covalent binding sites increased for diallyl disulfide. Based on these findings, it can be proposed that the covalent modification of ß-lactoglobulin functions as a specific transporter stabilizing allicin or diallyl disulfide.

ß-Lactoglobulin as nanotransporter--Part II: Characterization of the covalent protein modification by allicin and diallyl disulfide.

The whey protein ß-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified ß-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified ß-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to ß-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein.

Influence of fermentation on glucosinolates and glucobrassicin degradation products in sauerkraut.

A systematic investigation was carried out on the influence of fermentation on glucosinolates and their degradation products from fresh raw cabbage, throughout fermentation at 20 °C and storage at 4 °C. Glucosinolates were degraded dramatically between Day 2 and 5 of fermentation and by Day 7 there was no detectable amount of glucosinolates left. Fermentation led to formation of potential bioactive compounds ascorbigen (13.0 µmol/100 g FW) and indole-3-carbinol (4.52 µmol/100g FW) with their higher concentrations from Day 5 to Day 9. However, during storage indole-3-carbinol slowly degraded to 0.68 µmol/100 g FW, while ascorbigen was relatively stable from Week 4 until Week 8 at 6.78 µmol/100 g FW. In contrast, the content of indole-3-acetonitrile decreased rapidly during fermentation from 3.6 to 0.14 µmol/100 g FW. The results imply a maximum of health beneficial compounds after fermentation (7-9 days) in contrast to raw cabbage or stored sauerkraut.

Characterization of the covalent binding of allyl isothiocyanate to ß-lactoglobulin by fluorescence quenching, equilibrium measurement, and mass spectrometry.

Reversible binding of small compounds through hydrophobic interactions or hydrogen bonding to food proteins (e.g. milk proteins) is a thoroughly researched topic. In contrast, covalent interactions are not well characterized. Here, we report a rare form of positive-cooperativity-linear binding of allyl isothiocyanate with ß-lactoglobulin, resulting in the cleavage of a disulfide bond of the protein. We compared three methods (i.e. fluorescence quenching, equilibrium dialysis, and headspace-water equilibrium) to characterize the binding kinetics and investigated the molecular binding by mass spectrometry. The methodologies used were found to be comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium-based methods respectively.