Potential for Development of an Escherichia coli—Based Biosensor for Assessing Bioavailable Methionine: A Review

Methionine is an essential amino acid for animals and is typically considered one of the first limiting amino acids in animal feed formulations. Methionine deficiency or excess in animal diets can lead to sub-optimal animal performance and increased environmental pollution, which necessitates its accurate quantification and proper dosage in animal rations. Animal bioassays are the current industry standard to quantify methionine bioavailability. However, animal-based assays are not only time consuming, but expensive and are becoming more scrutinized by governmental regulations. In addition, a variety of artifacts can hinder the variability and time efficacy of these assays. Microbiological assays, which are based on a microbial response to external supplementation of a particular nutrient such as methionine, appear to be attractive potential alternatives to the already established standards. They are rapid and inexpensive in vitro assays which are characterized with relatively accurate and consistent estimation of digestible methionine in feeds and feed ingredients. The current review discusses the potential to develop Escherichia coli-based microbial biosensors for methionine bioavailability quantification. Methionine biosynthesis and regulation pathways are overviewed in relation to genetic manipulation required for the generation of a respective methionine auxotroph that could be practical for a routine bioassay. A prospective utilization of Escherichia coli methionine biosensor would allow for inexpensive and rapid methionine quantification and ultimately enable timely assessment of nutritional profiles of feedstuffs

Multifunctionality of rapeseed meal protein isolates prepared by sequential isoelectric precipitation

Rapeseed meal is a by-product of the oil-producing industry with a currently underesti-mated application. Two protein isolates, PI2.5–8.5 or PI10.5–2.5, were obtained from industrial rapeseed meal after treatment with an aqueous ethanol solution. The alkaline-extracted proteins were sequen-tially precipitated by two different modes, from pH 10.5 to 2.5, and vice versa, from 2.5 to 8.5, with a step of 1 pH unit. The preparation approach influenced both the functional and antioxidant properties of the isolates. The PI10.5–2.5 exhibited higher water and oil absorption capacities than PI2.5–8.5, reaching 2.68 g H2O/g sample and 2.36 g oil/g sample, respectively. The emulsion stability of the PI2.5–8.5, evaluated after heating at 80 °C, was either 100% or close to 100% for all pH values studied (from 2 to 10), except for pH 6 where it reached 93.87%. For the PI10.5–2.5, decreases in the emulsion stability were observed at pH 8 (85.71%) and pH 10 (53.15%). In the entire concentration range, the PI10.5–2.5 exhibited a higher scavenging ability on 2,2-diphenyl-1-picryl hydrazyl (DPPH) and hydroxyl radicals than PI2.5–8.5 as evaluated by DPPH and 2-deoxyribose assays, respectively. At the highest concentration studied, 1.0%, the neutralization of DPPH radicals by PI10.5–2 reached half of that exhibited by synthetic antioxidant butylhydroxytoluene (82.65%). At the same concentration, the inhibition of hydroxyl radicals by PI10.5–2 (71.25%) was close to that achieved by mannitol (75.62%), which was used as a positive control. Established antioxidant capacities add value to the protein isolates that can thus be used as both emulsifiers and antioxidants.info:eu-repo/semantics/publishedVersio

Enhanced solubility of rapeseed meal protein isolates prepared by sequential isoelectric precipitation

The solubility of plant protein isolates is a key determinant of their potential application. Two protein isolates (PI) from ethanol-treated industrial rapeseed meal, PI10.5–2.5 and PI2.5–8.5, were prepared by sequential isoelectric precipitation of alkali-extracted proteins (pH 12) starting from pH 10.5 to 2.5 or from pH 2.5 to 8.5, respectively. Biochemical analyses revealed that PI2.5–8.5 contained a higher amount of crude protein (72.84%) than PI10.5–2.5 (68.67%). In the same protein isolate, the level of total phenols (0.71%) was almost two-fold higher than that in PI10.5–2.5 (0.42%). No glucosinolates were established in both protein isolates. SDS-PAGE analysis demonstrated that PI10.5–2.5 contained 10 to 15 kDa protein fractions in a relatively higher amount, while PI2.5–8.5 was enriched in 18 to 29 kDa protein fractions. PI10.5–2.5 exhibited high solubility, varying from 41.74% at pH 4.5 to 65.13% at pH 6.5, while PI2.5–8.5 was almost two-fold less soluble under the same conditions. Up to pH 5.5, the addition of NaCl at 0.03 and 0.25 M diminished the solubility of PI2.5–8.5, while the solubility of PI10.5–2.5 was increased. The supplementation of PI10.5–2.5 with 0.25 M NaCl enhanced the protein solubility to 56.11% at pH 4.5 and 94.26% at pH 6.5. The addition of 0.03 M NaCl also increased the solubility of this protein isolate but to a lower extent. Overall, the approach for sequential precipitation of proteins influenced the biochemical characteristics, protein fractional profile and solubility of prepared protein isolates.info:eu-repo/semantics/publishedVersio

Freeze and freeze drying applied as preservation processes of melon peel

info:eu-repo/semantics/submittedVersio

Functional properties of protein isolate and acid soluble protein-rich ingredient co-produced from ethanol-treated industrial rapeseed meal

Rapeseed meal is produced in large quantities as a by-product of vegetable oil production. To enhance the utility and profi tability of the rapeseed meal, it was treated with ethanol and used for concomitant preparation of two protein-rich ingredients, namely protein isolate (PI) and acid soluble protein (ASP). Their functional properties were evaluated in response to two boundary concentrations of NaCl (0.03 and 0.25 mol/L) in a wide pH range (2 to 10). The PI exhibited the lowest protein solubility at isolectric point (pH 4.5) which increased both at lower and higher pH. In contrast, ASP exhibited high protein solubility (>70%) which was negligibly influenced by pH. The addition of 0.03 mol/L NaCl increased its protein solubility to almost 100% at acidic pH. The water holding capacity of PI was positively influenced by the addition of 0.25 mol/L NaCl. The ASP did not exhibit any capacity to hold water but demonstrated higher ability to absorb oil compared to the PI. Both ingredients exhibited different thermal stability in response to salt addition at pH 7 and 8. PI and ASP exhibited completely different pattern of emulsion stability as influenced by pH. While the stability of PI emulsions was close to 100% and only negligibly affected by pH, the ASP emulsion stability significantly varied in response to pH variation. The concomitant production of PI and ASP resulted in products with distinctive techno-functional properties, which makes them suitable for different applications as additives in the formulation of new food products.info:eu-repo/semantics/publishedVersio

Pepsin-Assisted Transglutaminase Modification of Functional Properties of a Protein Isolate Obtained from Industrial Sunflower Meal

Upotrebom industrijske suncokretove sačme za dobivanje proizvoda bogatih proteinima moguće je adekvatno i učinkovito iskoristiti taj poljoprivredni nusproizvod. Iako proteini suncokretove sačme imaju specifična svojstva, potrebno ih je poboljšati da bi se povećala mogućnost njihove primjene za dobivanje proizvoda visoke prehrambene vrijednosti. Svrha je ovoga rada bila ocijeniti zajednički učinak djelomične hidrolize pomoću pepsina i modifikacije transglutaminazom na funkcionalnost izolata industrijske suncokretove sačme pri pH- -vrijednostima od 2 do 10. Dobivena su tri hidrolizata pepsina modificirana pomoću transglutaminaze, nazvani TG-PH1 (stupanj hidrolize od 0,48 %), TG-PH2 (stupanj hidrolize 0,71 %) i TG-PH3 (stupanj hidrolize 1,72 %). Sva su tri hidrolizata imala bolju topljivost pri pH-vrijednostima od 3,5 do 5,5; a najveću je topljivost imao izolat TG-PH3 pri izoelektričnoj točki proteina (pI=4,5). Izolat proteina suncokretove sačme te izolat suncokretove sačme modificiran pomoću transglutaminaze imali su bolju topljivost od tri hidrolizata modificirana pomoću transglutamnaze pri pH7. Primjenom sva tri hidrolizata pepsina bitno je poboljšana sposobnost stvaranja pjene (p<0.05) pri cijelom ispitanom rasponu pH-vrijednosti. Hidroliza izolata proteina pomoću pepsina s tri različita stupnja hidrolize nije poboljšala stabilnost pjene. Hidrolizat TG-PH3 imao je bolju toplinsku stabilnost, i to do 80 °C, u usporedbi s izolatom proteina (pH=7). Pri 90 °C, najveća je toplinska stabilnost postignuta modifikacijom izolata proteina pomoću pepsina. Obradom izolata proteina transglutaminazom nakon hidrolize pomoću pepsina mogu se proizvesti izolati suncokretove sačme s poboljšanom topljivošću, sposobnošću stvaranja pjene i povećanom toplinskom stabilnosti za primjenu u prehrambenoj industriji.The utilization of industrial sunflower meal to produce protein-rich products for the food industry is an alternative approach for better and more efficient use of this agricultural by-product. Sunflower meal proteins possess specific functional properties, which however need improvement to broaden their potential as supplements for delivering high-quality products for human nutrition. The aim of the study is to evaluate the combined influence of low-degree pepsin hydrolysis and transglutaminase (TG) modification on industrial sunflower meal protein isolate functionality at pH=2 to 10. Three TG-modified pepsin hydrolysates with the degree of hydrolysis of 0.48, 0.71 and 1.72 % were produced and named TG-PH1, TG-PH2 and TG-PH3, respectively. All three TG-modified pepsin hydrolysates exhibited improved solubility at pH between 3.5 and 5.5 as the highest was observed of TG-PH3 at protein isoelectric point (pI=4.5). Sunflower meal protein isolate and TG-modified sunflower meal protein isolate had greater solubility than the three TG-modified hydrolysates at pH7. Significant improvement of foam making capacity (p<0.05) was achieved with all three TG-modified pepsin hydrolysates in the entire pH area studied. Pepsin hydrolysis of the protein isolate with the three degrees of hydrolysis did not improve foam stability. Improved thermal stability was observed with TG-PH3 up to 80 °C compared to the protein isolate (pH=7). At 90 °C, TG modification of the protein isolate alone resulted in the highest thermal stability. Pepsin hydrolysis followed by a treatment with TG could be used to produce sunflower protein isolates with improved solubility, foam making capacity and thermal stability for use in the food industry

Effect of non-thermal processing on the aromatic profile of Cantaloupe melon juice

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Growth and transcriptional response of Salmonella Typhimurium LT2 to glucose-lysine-based Maillard reaction products generated under low water activity conditions

El pdf del artículo es la versión pre-print.-- et al.This study reports the growth and transcriptional responses of Salmonella Typhimurium LT2 to Maillard reaction products (MRPs) generated in a simulation of the reaction during food processing and storage. Maillard reactions between N- α-acetyl-lysine and glucose were stopped at 4, 23, or 143. h to generate sub-samples with prevailing Amadori compound, advanced glycation end products (AGEs), or melanoidins, respectively. When used as the only carbon source, MRPs' disappearance occurred during S. Typhimurium secondary logarithmic growth phase after the glucose available in the MRP reaction mixtures was exhausted. Of the three MRP sub-samples, the Amadori compound was the preferred carbon source (98% disappearance). Decreases in AGEs (37%) and melanoidins (15%) also occurred. Transcription profiles of cells grown on the MRPs revealed predominant up-regulation of genes associated with the functional groups of energy metabolism, fatty and phospholipid metabolism, cellular process, and regulatory functions, and general down-regulation of the genes in the groups of amino acid biosynthesis, protein synthesis and transcription, transport and binding proteins, and DNA metabolism. © 2011 Elsevier Ltd.This research was supported by a USDA-CREES Food Safety Consortium grant, a USDA/NIFSI grant number 0403 90950-24-2192, the Consolider Ingenio 2010 Programme FUN-C-FOOD CSD2007-00063 (Ministerio de Educación y Ciencia) project and the ANALISYC II S2009/AGR-1464 (Comunidad de Madrid). O. Hernandez-Hernandez thanks CSIC for a JAE Predoctoral grant.Peer Reviewe

Development of rapid microbial methods for lysine quantification in feed ingredients based on green fluorescent protein fluorescence

Lysine is one of the more limiting amino acids in protein sources for chickens. Since lysine is also an essential amino acid for animals, it is an important component of animal dietary formulation. Therefore, an accurate pre-determination of bioavailable lysine in feedstuffs is important. An optical density (OD) based microbiological assay for lysine determination using E. coli lysine auxotroph has been previously developed. However, because the assay is based on bacterial growth response to extracellular lysine measured as OD, it can be relatively time consuming (10-12h). Therefore, more rapid assays are needed if pre-formulation estimates are required. In this dissertation whole cell fluorescent biosensors for the quantification of bioavailable and total lysine in feed protein sources were developed. The biosensor for quantification of bioavailable lysine was based on the growth response of E. coli to an external source of lysine and lysinecontaining small peptides. Green fluorescent protein (GFP) was inserted in the genome of E. coli lysine auxotroph as a part of a mini-Tn5- transposon by conjugation. Bacterial growth response to external lysine and small peptides was monitored and recorded by measuring the fluorescence emitted by GFP. The second type biosensor developed was designed for the quantification of total lysine. It was based on the measurement of a promoter activity, which was induced and modulated by extracellular concentration of lysine. Cad promoter was amplified from E. coli K-12 genome and was cloned into promoterless gfp plasmid. The construct was electroporated into electrocompetent E. coli cells. The promoter activity was induced under the conditions of low pH and graded concentrations of lysine. Lysine-dose response was measured by the fluorescence of GFP. Both methods were characterized as having a high potential for practical application