Generation of a green fluorescent protein gene chromosomal insertion containing Escherichia coli strain for gene induction-based quantification of bioavailable lysine

The importance of lysine determination in feed materials is crucial for the feed industry because this amino acid can be limiting in many of the cereal materials used for animal feeds. The bacterial gene induction-based assay developed in this study aimed to measure lysine bioavailability in feeds as an alternative analytical method for animal assays. The advantages of a gene induction-based approach include rapid and quantitative estimation of many samples and results that relate a bacterial response to a biological response observed in animals. A whole-cell biosensor strain was constructed using a fluorescent E. coli strain that has an inducible fluorescent phenotype sensitive to extracellular lysine contents. A genetic fusion that links the promoter of cad operon with a green fluorescent protein encoding gene (gfp) was constructed, and a fluorescent assay was developed. A standard lysine curve (R² = 0.95) was generated and used for lysine bioavailability quantification of four feed ingredients (whole egg protein, blood-, soybean-, and meat and bone meal). Quantities as low as 50 μg/ml protein of digested samples were sufficient for analyses using the biosensor, except for meat and bone meal. Because of the low levels of free lysine in non-digested samples, fluorescence of these protein sources containing lower than 500 μg/ml protein was not detected (except for soybean meal). The results using enzymatically digested protein sources showed that the test strain emitted a fluorescent response that was proportional to the level of lysine present in the feed samples.     

Antibacterial activity of proteins extracted from the pulp of wild edible fruit of Bromelia pinguin L.

Bromelia pinguin L. is a natural source of bioactive compounds. The main purpose of this research was to isolate and characterize bioactive proteins from its fruit. B. pinguin proteins were fractionated by gel filtration chromatography, and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antibacterial activity of the proteins was analyzed against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, and the enzymatic activity was evaluated by protease activity and trypsin inhibitions assays. Protein fraction obtained by gel filtration chromatography exhibited antibacterial activity against E. coli (minimum inhibitory concentration [MIC] 0.3492 mg/mL) and S. aureus (MIC 0.6845 mg/mL). The proteolytic activity of the fraction was 0.985 U_cas/mL. The substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay detected protease inhibitors with molecular weights of 43 and 74 kDa. Antibacterial studies of E.coli and S. aureus were determined by comparing the protein fraction with different antibiotics. The antibacterial activity of proteins extracted from the pulp of the fruit of Bromelia pinguin L. could be related to the presence of enzymes, protease inhibitors and peptides.     

Availability of amino acids in processed plant-protein foodstuffs

The microbiological availability of 6 essential amino acids in some processed plant protein foodstuffs was assessed with different micro-organisms after digestion with different proteolytic enzymes (papain, pepsin, trypsin, erepsin) or their combinations. Available lysine was also determined with the dinitrofluoro-benzene (DNFB) procedure. The amounts of ‘available’ amino acids were compared with those liberated by supposedly complete hydrolysis. More lysine was found ‘available’ with the DNFB method than with Leuconostoc mesenteroides following digestion with pepsin and trypsin. Digestion with papain released less methionine and tryptophan available for Streptococcus zymogenes than did digestion with pepsin. Treatment with trypsin after digestion with pepsin often increased the liberation of amino acids, whereas digestion with erepsin had little or no additional effect. Methionine and tryptophan were assessed by two procedures, but different results were obtained with different protein sources. Continuous digestion-dialysis did not liberate more lysine, methionine and tryptophan than non-dialysed digests.     

THE PURIFICATION OF PROTEASE FROM COWSLIP (PRIMULA VERIS) AND ITS USE IN FOOD PROCESSING

Proteases perform very important functions and are used in different objectives as in vitro and in vivo. In recent years, proteases have been used for clinical, pharmaceutical and industrial applications. Most of these proteases are obtained from animal sources. News of diseases passing over from animals to human (severe acute respiratory syndrome [SARS], mad cow) has created suspicion of contamination in animal food and its product. Therefore, a new vegetal protease source for use in the food industry was studied. Ammonium sulfate fractionation and CM‐sephadex ion exchange chromatography were used in the purification of the enzyme. The purified enzyme has an optimum pH of 7.0 and its optimum temperature was 70C. The V_max and K_m values determined by Lineweaver–Burk graphics were 0.44 mg/mL.min and 40 mM, respectively. The purification degree was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE ). The native molecular weight of the enzyme was 46 kDa using a gel filtration chromatograph. SDS‐PAGE results show that the enzyme has two subunits, which have protease activity at 32 and 14 kD. It was investigated whether the purified and characterized protease enzyme would cause to congeal milk or could produce digestion of chicken and cow meat. The results showed that the protease enzyme can be used for industrial production.     

Relationship between proportion and composition of albumins,and in vitro protein digestibility of raw and cooked pea seeds (Pisum sativum L.)

BACKGROUND: Peas provide an excellent plant protein resource for human diets, but their proteins are less readily digestible than animal proteins. To identify the relationship between composition and in vitro digestibility of pea protein, eight pea varieties with a wide range of protein content (157.3–272.7 g kg^?1) were determined for the proportion of albumins and globulins, their compositions using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and in vitro protein digestibility (IVPD) before and after heat treatment using a multi‐enzyme (trypsin, chymotrypsin and peptidase) method. RESULTS: The proportion of albumins based on total seed protein content decreased from 229 to 147 g kg^?1 as seed protein content increased from 157.3 to 272.7 g kg^?1, while the proportion of globulins increased from 483 to 590 g kg^?1. The IVPDs of eight raw pea seeds were 79.9–83.5%, with significant varietal variations, and those were improved to 85.9–86.8% by cooking. Albumins, including (pea albumins 2) PA2, trypsin inhibitor, lectin and lipoxygenase, were identified as proteolytic resistant proteins. Globulins were mostly digested by protease treatment after heating. CONCLUSION: The quantitative ratio of albumins and globulins, and the quantitative variations of albumin protein components, including lipoxygenase, PA2, lectins and trypsin inhibitors, appear to influence the protein digestibility of both raw and cooked pea seeds. Copyright © 2010 Society of Chemical Industry     

D-Lysine,alloisoleucine and lysinoalanine in supplementary proteins with different lysine availabilities

D -Lysine, alloisoleucine and lysinoalanine were determined in 16 commercial protein supplements on which lysine availability had been measured by slope-ratio assay with pigs and by chemical dinitrophenylation. About 2.5% of lysine was racemised by protein hydrolysis in 6 M HCl. Only three of 10 samples with poor lysine availability by slope-ratio assay contained significantly more D -iysine than control proteins (P < 0.01). D -Lysine was not significantly correlated with lysine availability by either method; nor did it improve the poor correlation between the slope-ratio assay and dinitrophenylation. The highest level of alloisoleucine was less than 14% that of isoleucine. In all proteins except skim milk powder lysinoalanine occurred at 0.3% or less of the corresponding lysine level. Neither alloisoleucine nor lysinoalanine was related to lysine availability.     

Heterologous production of basidiomycetous peptidases to decrease sodium chloride in food

In the past, the topic of excessive salt consumption associated with various chronic diseases became a focus of nutritional science. Enzymes such as hydrolases and specifically peptidases are widely used in industry since they do not require cofactors, are often stable at high pH values, and have a broad cleavage specificity. Thus, a new approach to use peptidases would be the generation of L-arginyl dipeptides, which do not have a taste on their own, but are able to enhance the salty taste. This thesis should focus on the isolation and characterisation of arginyl-specific peptidase genes from basidiomycetes to generate these dipeptides. First, various peptidase genes from Trametes versicolor, Phanerochaete chrysosporium and Schizophyllum commune were isolated and expressed in Escherichia coli or Komagataella phaffii. The serine (ABB73029) peptidase from P. chrysosporium and the aspartate peptidase from T. versicolor (EIW62808) were successfully purified. Activity assays showed that the aspartate peptidase from T. versicolor is not an arginyl-specific peptidase, while the analysis of cleavage sites indicates that the serine peptidase from P. chrysosporium is an exopeptidase. Next, 29 basidiomycetes were cultivated over 24 days in minimal medium with 1 % gluten and analysed regarding their endo- and exopeptidase activity. Out of 29 fungi, five interesting species with arginyl-specific peptidase activity were chosen, namely Agrocybe aegerita, Fomitop- sis pinicola, Flammulina velutipes, Hypholoma sublateritium and Pleurotus eryngii. After purification of the culture supernatant using size exclusion chromatography, active fractions were used to hydrolyse the substrate casein, and cleavage products were analysed. Cleavage sites were only detected for A. aegerita, F. velutipes and P. eryngii. Beside arginyl-specific cleavage sites also a high amount of undesired exopeptidase cleavage sites was determined assuming that these peptidases are probably exopeptidases rather than arginyl-specific peptidases. Generation of salt taste enhancing dipeptides should finally be achieved with a dipeptidyl- peptidase V (DPPV) from Pleurotusfloridanus. PflDPPV was heterologously produced in E. coli, had optima at pH 8.5 and 60 °C and was not completely inhibited by the tested peptidase inhibitors.     

Growth Inhibitory Effect of Chicken Egg Yolk Antibody (IgY) on Escherichia coli O157:H7

Escherichia coli O157:H7‐specific antibodies (immunoglobulin Y [IgY]) were isolated by the water‐dilution method from the egg yolk of chickens that were immunized with E. coli O157:H7 whole cells. The specific‐binding activity of IgY against E. coli O157:H7 as determined by the enzyme immuno assay showed high levels of activity against bacterial whole cells. IgY binding activity was further demonstrated to have an inhibitory effect on E. coli O157:H7 growth in a liquid medium. The antibacterial function of IgY appeared to result from the interaction of IgY with surface components of E. coli O157:H7, as proven from observation of immunofluorescence and immunoelectron microscopy.     

SPECIFICITY OF ESCHERICHIA COLI LYSINE AUXOTROPH GROWTH KINETICS AND LYSINE ASSAY RESPONSE TO VARIOUS SOLUBLE MAILLARD REACTION PRODUCTS

Lysine availability in foods or feeds can be rapidly assessed by an Escherichia coli lysine auxotroph (lys) based assay. The overall objective of these studies was to examine if the accuracy of this lysine assay was affected by the presence of different soluble Maillard reaction products (MRP) in a model system. The standard curve without addition of MRP was parallel to the standard curves with addition of histidine-MRP (histidine with glucose) or arginine-MRP (arginine with glucose). The y-axis intercept of the standard curve without addition of MRP was significantly (P < 0.05) greater than the y-axis intercepts of the standard curves with addition of MRP. This data indicated that MRP inhibited lys growth when limited by lysine concentration, and accurate determination of lysine availability in food or feed sample may require an internal standard curve where known lysine increments are added to the samples to be analyzed.     

The DNA Sequence of the Escherichia coli O22 O-Antigen Gene Cluster and Detection of Pathogenic Strains Belonging to E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and Genes in the Respective O-Antigen Gene Clusters

Multiplex polymerase chain reaction (PCR) assays were developed for detection of pathogenic strains belonging to Escherichia coli serogroups O22 and O91. The O-antigen gene cluster of E. coli O22 was sequenced to identify genes that could be employed as targets for serogroup-specific PCR assays. The wzx and wzy genes in the O-antigen gene clusters of E. coli O22 and E. coli O91 were selected as target genes. The assays were serogroup-specific when tested against 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, humans, and animals, representative strains belonging to 168 E. coli O serogroups and non-E. coli bacteria. Furthermore, 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, water, animals, and humans were tested by the PCR for the presence of six and 19 virulence genes, respectively, associated with pathogenic E. coli strains. Based on the PCR screening results, multiplex PCR assays targeting the O22 wzy gene and the cnf-1 and sfa genes in E. coli O22 and the O91 wzy gene, conserved sequences of stx ₁ and stx ₂ genes, and the astA and cdt-III genes in E. coli O91 were developed to detect and identify pathogenic strains belonging to serogroups O22 and O91. Furthermore, E. coli O22 and O91 were detected by multiplex PCR assays targeting the wzx or wzy genes and conserved sequences of the stx ₁ and stx ₂ genes in ground beef samples inoculated with approximately two colony-forming units (CFU)/25 g after 18-h enrichment. The results demonstrate that the E. coli O22 and O91 wzx and wzy gene sequences were specific for the respective serogroups and can be used as diagnostic markers for rapid identification of these serogroups as an alternative to serotyping. The multiplex PCR assays targeting the O22 and O91 wzx and wzy genes and virulence genes can be used to identify and to detect pathogenic strains of these serogroups in food and fecal samples. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Activity and bioavailability of food protein-derived angiotensin-I-converting enzyme–inhibitory peptides

Angiotensin-I-converting enzyme (ACE) inhibitory peptides are able to inhibit the activity of ACE, which is the key enzymatic factor mediating systemic hypertension. ACE-inhibitory peptides can be obtained from edible proteins and have the function of antihypertension. The amino acid sequences and the secondary structures of ACE-inhibitory peptides determine the inhibitory activities and stability. The resistance of ACE-inhibitory peptides to digestive enzymes and peptidase affect their antihypertensive bioactivity in vivo. In this paper, the mechanism of ACE-inhibition, sources of the inhibitory peptides, structure–activity relationships, stability during digestion, absorption and transportation of ACE-inhibitory peptides, and consumption of ACE-inhibitory peptides are reviewed, which provide guidance to the development of new functional foods and production of antihypertensive nutraceuticals and pharmaceuticals.     

Characterization of L-Arabinose Isomerase in Bacillus subtilis,a GRAS Host,for the Production of Edible Tagatose

L-Arabinose isomerase (AI; E.C. 5.3.1.4), a commercial enzyme for the production of edible tagatose in vitro and ribulose production in vivo, has been studied using enzymes expressed in an Escherichia coli system, which might cause noxious by-products in food. To ensure food safety in the tagatose manufacturing process, we studied an AI expression system in Bacillus subtilis. The AI gene from Geobacillus stearothermophilus (GSAI) was expressed in Bacillus subtilis, a GRAS host used in the production of fermented soybean in Korea, after subcloning into a Bacillus subtilis - E. coli shuttle vector, and was characterized after purification. The activities of the crude enzyme extract and a purified sample were 0.15 U/mg protein and 2.7 U/mg protein, respectively. The optimal pH and the optimal temperature for arabinose and galactose as substrates were pH 8.0 and 60°C, respectively, the same as those for GSAI in an E. coli expression system. Substrate affinities (K_m) for arabinose and galactose were 77 mM and 279 mM, respectively, whereas in the E. coli expression system, they were 100 mM and 578 mM, respectively. Catalytic efficiencies (k_cat/K_m) for arabinose and galactose were 58.3 and 11.4 mM^?1 min^?1, respectively. The potential use of GSAI expressed in a GRAS host for the production of edible tagatose is discussed in light of these results.     

Antibacterial activity and mechanism of a novel bacteriocin produced by Lactiplantibacillus plantarum against Escherichia coli and Staphylococcus aureus

The purpose of the study was to explore the antibacterial effect and mechanism of a novel bacteriocin of Lactiplantibacillus plantarum (L. plantarum) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The antibacterial activity was determined by the Oxford cup method, and the dynamic growth curves were conducted with continuous shaking incubation. The result showed that the bacteriocin was a protein or protein-like antibacterial substance susceptible to pepsin and trypsin. And it had good antibacterial activity, pH stability, thermostability and enzyme treatment stability against E. coli and S. aureus. The SEM, flow cytometry and nucleic acid leakage showed that the bacteriocin disrupted the cell structures of the two bacteria by damaging cell walls and cell membranes. An agarose gel electrophoresis and SDS-PAGE analysis showed that the bacteriocin inhibited DNA replication and interfered with the protein formation, which resulted in the inhibition of the two bacteria's growth. Therefore, the use of the L. plantarum bacteriocin might be a promising biocontrol strategy to inhibit the pollution of E. coli and S. aureus simultaneously in foods.     

Removal of Microbial Biofilms from Dispense Equipment: The Effect of Enzymatic Pre‐digestion and Detergent Treatment

Microbial biofilms can form in dispense outlets as a result of poor or inadequate cleaning and can be difficult to remove using conventional practices. Enzymatic cleaners might help to remove biofilms by degrading the exopolysaccharide layers in which the microbes are embedded. A multispecies biofilm comprising wild type dispense isolates of Flavimonas oryzihabitans, Lactobacillus brevis, Leuconostoc mesenteroides and Saccharomyces cerevisiae was generated on a section of tubing and fitted into a pilot dispense system, which was left uncleaned for 12 weeks. After cleaning approximately 10⁴ viable aerobes and 10³ viable anaerobes were still present. Stainless steel coupons and pieces of dispense line contaminated with biofilm were incubated in the laboratory with an enzyme mix containing varying proportions of α‐amylase, β‐glucuronidase, glucose oxidase, dextranase, protease and pectinase. Cultures grown on stainless steel had significantly (F pr. > 0.05) less viable cells than non‐enzyme treated biofilms, but this was dependent on the microbial species. Typically Lactobacillus brevis was most susceptible to the enzyme treatment. Cultures grown on dispense line were much more resistant to enzymatic digestion. Pre‐digestion with protease was most effective for removal of Lactobacillus brevis and Leuconostoc mesenteroides but not for Saccharomyces cerevisiae and Flavimonas oryzihabitans. In the simulated bar, pre‐digestion with protease reduced the viable cell count by 0.64 log units for the aerobes and 1.9 log units for the anaerobes. This study demonstrates that pre‐digestion with enzyme solutions before line cleaning is useful for treating heavily contaminated lines in trade.     

Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.     

Comparison of Lupinus angustifolius protein digestibility in dependence on protein,amino acids,trypsin inhibitors and polyphenolic compounds content

Lupine seeds are proposed as a source of valuable protein in human and animal nutrition. However, not only protein content but its bioavailability is important. Thus, the protein digestibility of twenty Lupinus angustifolius varieties seeds and the impact of some factors (protein and polyphenols content, trypsin inhibition and amino acids composition) on the protein digestion were discussed. Composition as well as the digestibility of these seeds was influenced by variety, weather conditions (precipitations) and place of cultivation (soil quality). The average digestibility of the seeds was 46%. The positive impact on the digestibility had the extractivity of the protein. Higher than it was expected activity of trypsin inhibitors (even up to ~32%) in some varieties influenced the protein availability, while the lysine and phenolic compounds content did not affect digestibility. The content of compounds influencing protein digestibility depends both on variety as well as on agronomical conditions.     

A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products

A 7-plex PCR assay was developed to achieve an effective detection and identification of serotype Enteritidis of Salmonella spp. and shiga toxin-producing type of Escherichia coli O157 in meat products. Six DNA sequences in the invA and sdfI genes of Salmonella Enteritidis as well as rfbE, eae, stx1, and stx2 genes of E. coli O157:H7 were employed to design primers. The multiplex PCR assay could specifically and sensitively detect and identify target pathogens. Applying the assay to meat samples, the multiplex PCR assay was able to simultaneously detect and identify the two pathogens at a sensitivity of three CFU/10 g raw meats after simple 16 h enrichment in buffered peptone water. Further applying in 21 retail meat samples revealed that two samples were positive for non-shiga toxin producing E. coli O157, one sample was positive for Stx2 producing E. coli O157 and five samples were positive for Salmonella enterica Enteritidis. Taken together, the 7-plex PCR assay is a rapid and reliable method for effectively screening single or multiple pathogens occurrences in various meat products, and could also identify the Salmonella enterica Enteritidis from all Salmonella spp. and shiga toxin producing type from all E. coli strains. Considering as a non expensive screening tool, the multiplex PCR assay has a great potential in complement for food stuff analysis by conventional microbiological tests.     

ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

Nutritive value of meat meals. II. Influence of raw materials and processing on protein quality

Commercial meat meals have shown low net protein utilisation values with rats of about 32, which is only 40% of the corresponding value for freeze-dried meat or gut. Biological assays for methionine and tryptophan, based on the weight gains of chicks, have shown a similar inferiority of meat meals despite 81–87% digestibility of their N. It is concluded that the lysine, methionine and tryptophan of the meat meals are approximately 80% available and that dilution of muscle protein with tendon and ossein in the raw material is at least as important as processing damage in reducing the quality of the products. It is argued that previous estimates of only 30% availability of methionine in meat meals are erroneous. Further problems in the use of values for fluorodinitrobenzene-available lysine and Streptococcus zymogenes-ayailable trytophan are discussed.     

STABILITY OF A PANCREATIC ENZYME COCKTAIL DURING IN VITRO PROTEIN DIGESTIBILITY ASSAYS

To maximize the efficiency of utilization of a pancreatic enzyme cocktail and estimate the contamination for in vitro protein digestibility assays, the specific activity losses of trypsin and chymotrypsin and the digestion of the enzyme proteins were studied. In the absence of protein substrate, increase of enzyme concentration augmented the half‐lives of trypsin and chymotrypsin and decreased the digestion of enzymatic proteins. In contrast, in the presence of substrate, increase of enzyme concentration decreased trypsin's half‐life. Increase of pH augmented the digestion of enzymatic proteins. The results indicated the optimum time for utilization of the enzymes depended on pH, enzyme concentration and presence of substrate. At the time when digestion of the proteins ceased, the average size of the hydrolysates was calculated between 3.1 and 5.4 amino acid residues, suggesting most proteins in the enzyme cocktail would be detected as digestible proteins.