Application of an Escherichia coli green fluorescent protein-based lysine biosensor under nonsterile conditions and autofluorescence background

AIMS: To examine the utility of an Escherichia coli green fluorescent protein (GFP) containing biosensor for quantification of bioavailable lysine in selected feed samples under nonsterile conditions and to estimate the background fluorescence of analyzed feed samples and evaluate the risk of confounding GFP emission from the lysine assay organism. METHODS AND RESULTS: Escherichia coli lysine auxotroph GFP based biosensor was used to determine the percentage of bioavailable lysine in two samples of soybean-, cottonseed-, and meat and bone meal under nonsterile conditions. The fluorescence emitted by GFP was successfully measured using a spectrofluorimeter to monitor bacterial growth response to protein-derived lysine and lysine containing small peptides. The autofluorescence of analyzed feed samples at different concentrations could also be estimated. CONCLUSIONS: When feed protein concentrations are decreased, autofluorescence interference can be avoided. SIGNIFICANCE: The E. coli lysine auxotroph GFP-based biosensor can successfully be used for the determination of bioavailable lysine in these selected animal feed proteins under nonsterile conditions. IMPACT OF THE STUDY: E. coli GFP biosensor for lysine has potential for routine application in animal feeds.     

Inclusion of sorghum,millet and cottonseed meal in broiler diets: a meta-analysis of effects on performance

A meta-analysis was conducted (i) to evaluate broiler response to partial or total substitution of corn by sorghum and millet and (ii) to determine the effect of soybean meal replacement by cottonseed meal in broiler diet. The database included 190 treatments from 29 experiments published from 1990 to 2013. Bird responses to an experimental diet were calculated relative to the control (Experimental−Control), and were submitted to mixed-effect models. Results showed that diets containing millet led to similar performance as the corn-based ones for all parameters, whereas sorghum-based diets decreased growth performance. No major effect of the level of substitution was observed with millet or cottonseed meal. No effect of the level of substitution of sorghum on feed intake was found; however, growth performance decreased when the level of substitution of corn by sorghum increased. Cottonseed meal was substituted to soybean meal up to 40% and found to increase feed intake while reducing growth performance. Young birds were not more sensitive to these ingredients than older birds since there was no negative effect of these ingredients on performance in the starter phase. Results obtained for sorghum pointed out the necessity to find technological improvements that will increase the utilization of these feedstuffs in broiler diet. An additional work is scheduled to validate these statistical results in vivo and to evaluate the interactions induced with the simultaneous inclusions of sorghum, millet and cottonseed meal in broiler feeding.     

Development of a whole cell green fluorescent sensor for lysine quantification

As an essential amino acid, lysine is an important component of animal and human diets and its bioavailability can depend on a variety of factors. Therefore, an accurate pre-determination of bioavailable lysine in foods and feeds is important. In this study a whole cell fluorescent biosensor for the quantification of lysine in protein sources was constructed. A gene encoding for green fluorescent protein (GFPmut3) was introduced into an E. coli lysine auxotroph genome as a part of a mini-Tn5-Km transposon. The location of the transposon was determined and the growth kinetics of the newly constructed biosensor were examined. The transposon disrupted the ybhM gene, which encodes for the synthesis of a protein with an unknown function. No effect of the transposon’s location in the genome or the expression of gfp on bacterial growth rates was observed. Based on the fluorescence emitted by GFPmut3, a standard curve after 6-h growth of the strain was generated. A correlation coefficient of 0.95 was observed when the fluorescence method was compared to the conventional optical density (OD) growth-based lysine assay. Using the newly developed lysine fluorescent whole cell sensor we determined the total lysine in casein acid hydrolyzate (7.13 ± 0.34%). When lysine added to 12 μg/ml and 30 μg/ml of casein acid hydrolyzate was quantified, recoveries of 97 ± 1.65% and 103 ± 4.66% respectively were detected. The results suggest that the microbial assay using GFP fluorescence represents a promising alternative method for the potential estimation of lysine in protein sources.     

Natural occurrence of zeralenone in feeds and feedstuffs used in poultry and pig nutrition in colombia

A total of 200 samples of feedstuffs and mixed feeds used for poultry and pig nutrition in Colombia were analysed for zearalenone using a LC technique with a limit of detection of 20 μg/kg. Samples of grain sorghum, maize, processed soybean, rice meal, cottonseed meal, and poultry and pig feeds, representative of the Colombian production for the 1995–1996 harvest, were taken at feed manufacturing plants located in different cities of the country. Zearalenone was detected in 25 of 45 samples of sorghum, 2 of 33 samples of maize, 7 of 22 samples of rice meal, 9 of 17 samples of cottonseed meal, 11 of 30 samples of poultry feed and 6 of 16 samples of pig feed. Zearalenone was not detected in soybean or other feedstuff s analysed (wheat by- products, cassava meal, palm). Overall levels of zearalenone ranged from 29 to 3956 μg/kg, with a mean value of 436 μg/kg. Only one of the 6 positive samples of pig feed had a zearalenone concentration above 500 μg/kg, which is normally considered as the safe level for pigs.     

Simultaneous inclusion of sorghum and cottonseed meal or millet in broiler diets: effects on performance and nutrient digestibility

Two experiments were conducted to investigate the use of sorghum, cottonseed meal and millet in broiler diets and their interaction when they are used simultaneously. In Experiment 1, a corn-soybean meal control diet was compared with eight experimental treatments based on low tannin sorghum (S30, S45 and S60), cottonseed meal (CM15, CM40) or both ingredients included in the same diet (S30/CM40, S45/CM25 and S60CM15). Results showed that BW gain was not affected by the inclusion of sorghum or cottonseed meal. However, feed intake tended to be affected by the cereal type with the highest values with sorghum-based diets. Feed conversion ratio increased (P<0.001) with sorghum-based diets compared with the control diet, whereas a combination of cottonseed meal and sorghum in the same diet did not affect the feed conversion ratio. Significant differences (P<0.001) were observed in apparent ileal digestibility (%) of protein and energy with the cottonseed meal and sorghum/cottonseed meal-based diets having lower protein and energy digestibility compared with corn-based diets. In Experiment 2, a control diet was compared with six diets in which corn was substituted at 60%, 80% or 100% by either sorghum or millet and other three diets with simultaneous inclusion of these two ingredients (S30/M30, S40/M40, S50/M50). Single or combined inclusion of sorghum and millet resulted in similar feed intake and growth performance as the control diet. Apparent ileal digestibility of protein and energy was higher with millet-based diets (P<0.001). Total tract digestibility of protein in sorghum and millet-based diets tended to decrease linearly with the increasing level of substitution. Sorghum-based diets resulted in lower total tract digestibility of fat compared with millet and sorghum/millet-based diets (P<0.001). Higher total tract digestibility of starch were obtained with the control diet and millet-based diets compared with the sorghum-based treatments. Results of the two experiments suggest that broiler growth performance was not affected by the dietary level of sorghum, millet or cottonseed meal. Nutrient digestion can, however, be affected by these feed ingredients.     

Apparent digestibility of crude protein and gross energy in some plant and animal-based feedstuffs by Clarias isheriensis (Siluriformes: Clariidae) (Sydenham 1980)

Apparent digestibility coefficient (ACD) of dry matter, crude protein and gross energy of white fish meal, blood meal, poultry by-product meal, hydrolysed feather meal, shrimp head meal, meat and bone meal, soybean meal, cottonseed meal, peanut meal, yellow maize, corn starch, cassava meal, sorghum, rice bran and cocoa pod meal were determined for Clarias isheriensis (47.5–51.2 g) fed to satiation with diets containing each test feedstuff in a 30:70 mixture with a reference diet, and chromium oxide as an indicator using the dissection technique. ADC_protein values ranged from 50 to 93 % while ADC_energy ranged from 42 to 98 %. Protein and energy content of animal or plant feed-stuffs were equally digestible to C. isheriensis.     

Prevalence of Fusarium species of the Liseola section on selected Colombian animal feedstuffs and their ability to produce fumonisins

A total of 57 samples of feedstuffs commonly used for animal nutrition in Colombia (corn, soybean, sorghum, cottonseed meal, sunflower seed meal, wheat middlings and rice) were analyzed for Fusarium contamination. Fusarium fungi were identified at species level by means of conventional methods and the ability to produce fumonisins of the most prevailing species was determined. A total of 41 of the feedstuffs analyzed (71.9%) were found to contain Fusarium spp. Most contaminated substrates were corn (100%), cottonseed meal (100%), sorghum (80%), and soybean (80%). Wheat middlings and rice showed lower levels of contamination (40% and 20%, respectively), while no Fusarium spp. could be isolated from sunflower seed meal. The most prevalent species of Fusarium isolated were F. verticilliodes (70.8%), F.␣proliferatum (25.0%), and F. subglutinans (4.2%). All of them correspond to section Liseola.Production of fumonisins on corn by the isolated Fusarium was screened through liquid chromatography. Almost all strains of F. verticilliodes (97.1%) produced FB1 (5.6–25,846.4 mg/kg) and FB2 (3.4–7507.5 mg/kg). Similarly, almost all strains of F.␣proliferatum (91.7%) produced fumonisins but at lower levels than F.␣verticilliodes (FB1 from 6.9 to 3885.0 mg/kg, and FB2 from 34.3 to 373.8 mg/kg), while F. subglutinans did not produce these toxins. This is the first study in Colombia describing toxigenic Fusarium isolates from␣animal feedstuffs.     

Composition of amino acids in feed ingredients for animal diets

Dietary amino acids (AA) are crucial for animal growth, development, reproduction, lactation, and health. However, there is a scarcity of information regarding complete composition of “nutritionally nonessential AA” (NEAA; those AA which can be synthesized by animals) in diets. To provide a much-needed database, we quantified NEAA (including glutamate, glutamine, aspartate, and asparagine) in feed ingredients for comparison with “nutritionally essential AA” (EAA; those AA whose carbon skeletons cannot be formed by animals). Except for gelatin and feather meal, animal and plant ingredients contained high percentages of glutamate plus glutamine, branched-chain AA, and aspartate plus asparagine, which were 10–32, 15–25, and 8–14% of total protein, respectively. In particular, leucine and glutamine were most abundant in blood meal and casein (13% of total protein), respectively. Notably, gelatin, feather meal, fish meal, meat and bone meal, and poultry byproduct had high percentages of glycine, proline plus hydroxyproline, and arginine, which were 10–35, 9.6–35, and 7.2–7.9% of total protein, respectively. Among plant products, arginine was most abundant in peanut meal and cottonseed meal (14–16% of total protein), whereas corn and sorghum had low percentages of cysteine, lysine, methionine, and tryptophan (0.9–3% of total protein). Overall, feed ingredients of animal origin (except for gelatin) are excellent sources of NEAA and EAA for livestock, avian, and aquatic species, whereas gelatin provides highest amounts of arginine, glycine, and proline plus hydroxyproline. Because casein, corn, soybean, peanut, fish, and gelatin are consumed by children and adults, our findings also have important implications for human nutrition.     

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA as a lacZ fusion protein was selected in a dapA ^- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.     

Efficacy of virginiamycin and a commercially-available lactobacillus probiotic in swine diets

Eight feeding trials using 708 cross-bred pigs were conducted to evaluate the addition of virginiamycin and a commercially-available lactobacillus fermention product (probiotic) to starter, grower and finisher diets based on maize and soya bean meal. Virginiamycin was added at a level of 11 mg/kg of diet, and lactobacillus probiotic at 1 g/kg in starter diets and 500 mg/kg in grower and finisher diets. In the combined analysis of four starter trials, pigs fed on diets containing virginiamycin tended to eat more and grow faster than pigs fed on the control diet, but feed efficiency was not different. Furthermore, daily gain, daily feed intake and feed efficiency were not improved by the addition of lactobacillus probiotic to starter diets. In the combined analysis of a starter—grower—finisher and a grower—finisher trial, virginiamycin supplementation did not alter overall pig performance compared with control. Pigs fed on a positive control diet containing chlortetracycline, penicillin and sulfamethazine had a nonsignificantly greater daily gain and feed intake.Daily gain for the lactobacillus probiotic-fed pigs was lower (P < 0.05) than that of the negative controls, but daily feed intake and feed efficiency were not significantly different. In three growing—finishing trials in which virginiamycin and lactobacillus probiotic were compared in a 2 × 2 factorial arrangement of treatments, daily gain and feed intakes were greater (P < 0.01) for pigs given virginiamycin. The lactobacillus probiotic did not improve daily gain, feed intake or feed efficiency. Overall, no interaction between virginiamycin and lactobacillus probiotic occurred.     

Apparent digestibilities and growth experiments with tilapia (Oreochromis niloticus) fed soybean meal, cottonseed meal and sunflower seed meal

Apparent digestibility and growth experiments with three oil seed by-products, soybean meal, cottonseed meal, and sunflower seed meal, were conducted using tilapia ( Oreochromis niloticus ) with an initial average body weight of 93 ± 5 g and 64 ± 1 g, respectively. The diets were formulated on an isonitrogeneous and isoenergetic basis. During the experimental period the fish were fed 1% and 1.5% of their metabolic body weight (kg^0.8) daily in the digestibility experiment and the growth experiment, respectively. Water temperature was maintained at 26.5 ± 1°C. Apparent digestibilities of the components and the diets were determined using an indirect method, i.e. with HCl-insoluble ash as an indicator. Faeces collection was carried out using a sedimentation technique.
Results of the apparent digestibilities showed that the crude protein, crude fat, as well as the gross energy digestibilities of soybean meal (93.0%, 94.6%, and 77.2%, respectively), were better than those of cottonseed meal (79.4%, 83.2%, and 57.9%, respectively) and sunflower seed meal (89.8%, 82.9%, and 49.3%, respectively). With the exception of the crude protein digestibility, nutrient and gross energy digestibilities of the cottonseed meal were found to be better than that of sunflower seed meal.
Growth experiment results indicated that soybean meal diet groups had inferior growth performance and feed conversion in comparison to the fishmeal control diet or the other test diets at an inclusion level of 32% of the digestible protein. Fish fed cottonseed meal diet and sunflower seed meal diet showed a relatively better, but not significantly different performance than did the fishmeal control diet. Supplementation diets with lysine and methionine did not enhance fish performance.     

Metabolically engineered soybean seed with enhanced threonine levels: biochemical characterization and seed‐specific expression of lysine‐insensitive variants of aspartate kinases from the enteric bacterium Xenorhabdus bovienii

Threonine (Thr) is one of a few limiting essential amino acids (EAAs) in the animal feed industry, and its level in feed rations can impact production of important meat sources, such as swine and poultry. Threonine as well as EAAs lysine (Lys) and methionine (Met) are all synthesized via the aspartate family pathway. Here, we report a successful strategy to produce high free threonine soybean seed via identification of a feedback‐resistant aspartate kinase (AK) enzyme that can be over‐expressed in developing soybean seed. Towards this goal, we have purified and biochemically characterized AK from the enteric bacterium Xenorhabdus bovienii (Xb). Site‐directed mutagenesis of XbAK identified two key regulatory residues Glu‐257 and Thr‐359 involved in lysine inhibition. Three feedback‐resistant alleles, XbAK_T359I, XbAK_E257K and XbAK_E257K/T359I, have been generated. This study is the first to kinetically characterize the XbAK enzyme and provide biochemical and transgenic evidence that Glu‐257 near the catalytic site is a critical residue for the allosteric regulation of AK. Furthermore, seed‐specific expression of the feedback‐resistant XbAK_T359I or XbAK_E257K allele results in increases of free Thr levels of up to 100‐fold in R₁ soybean seed when compared to wild‐type. Expression of feedback‐sensitive wild‐type AK did not substantially impact seed Thr content. In addition to high Thr, transgenic seed also showed substantial increases in other major free amino acid (FAA) levels, resulting in an up to 3.5‐fold increase in the total FAA content. The transgenic seed was normal in appearance and germinated well under greenhouse conditions.     

Biosensor studies of the binding of extracellular matrix components with immobilized Escherichia coli O157:H7 and inhibition by polysulfated polysaccharides

Binding interactions of immobilized E. coli O157:H7 with collagen I, fibronectin, laminin and glucoaminoglycans were studied utilizing a surface plasmon resonance biosensor. A model system was developed to evaluate the inhibition of collagen-laminin binding on the E. coli sensor surface with polysulfated polysaccharides such as heparan sulfate and carrageenans. Results showed that carrageenans inhibited 71–99% while heparan sulfate inhibited 39–41% of collagen/laminin binding to E. coli sensor surface. These studies allowed a rapid assessment of compounds for carcass treatment to inhibit or detach pathogens from meat and poultry.     

Bacterial protein meal produced on natural gas replacing soybean meal or fish meal in broiler chicken diets

Abstract

The effects of replacing soybean meal or fish meal with 2, 4 or 6% bacterial protein meal (BPM) on growth performance, ileal digestibility of amino acids and sensory quality of meat, were examined using 630 broiler chickens. Weight gain from 7 – 32 days of age did not differ significantly among the treatments. Efficiency of feed conversion was increased when BPM replaced soybean meal, and abdominal fat deposition tended to decline. Feed conversion was not affected when BPM replaced fish meal. Amino acid digestibility was unaffected or improved when BPM replaced soybean meal, whereas replacement of fishmeal with BPM resulted in similar digestibility. Sensory quality of fresh thigh meat was similar among treatments, but for freeze-stored chest meat replacement of fish meal with BPM reduced off-odour and off-flavour and increased juiciness. It was concluded that 6% BPM can replace soybean meal or fish meal protein in broiler chicken diets.     

Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

Bacterial protein meal produced on natural gas replacing soybean meal or fish meal in broiler chicken diets

The effects of replacing soybean meal or fish meal with 2, 4 or 6% bacterial protein meal (BPM) on growth performance, ileal digestibility of amino acids and sensory quality of meat, were examined using 630 broiler chickens. Weight gain from 7-32 days of age did not differ significantly among the treatments. Efficiency of feed conversion was increased when BPM replaced soybean meal, and abdominal fat deposition tended to decline. Feed conversion was not affected when BPM replaced fish meal. Amino acid digestibility was unaffected or improved when BPM replaced soybean meal, whereas replacement of fishmeal with BPM resulted in similar digestibility. Sensory quality of fresh thigh meat was similar among treatments, but for freeze-stored chest meat replacement of fish meal with BPM reduced off-odour and off-flavour and increased juiciness. It was concluded that 6% BPM can replace soybean meal or fish meal protein in broiler chicken diets.     

Apparent digestion coefficients for dry matter, protein and essential amino acids in terrestrial ingredients for Pacific shrimp Litopenaeus vannamei (Decapoda: Penaeidae)

Protein quality mainly depends on the essential amino acid (EAA) profile, but also on its bioavailability, because EAA digestibility is generally lower than the analyzed amounts. This information is needed in the aquaculture industry for aquafeed formulation. For this purpose, the apparent digestibility coefficients of dry matter, protein, and essential amino acids of eight feedstuffs of terrestrial origin were determined for the juvenile whiteleg shrimp Litopenaeus vannamei (15-19 g), using 1% chromic oxide as an inert marker. A reference diet was formulated and produced in the laboratory. Eight experimental diets were prepared each with 30% of one of the experimental ingredients added to the reference diet: casein, porcine byproduct meal poultry byproduct meal, corn meal, wheat gluten meal, soybean paste, sorghum meal, and wheat meal. The experiment consisted of a single-factor, completely randomized design with three replicates per treatment. Samples of ingredients, diets and feces were analyzed for nitrogen and amino acids. For amino acid assay, we used reverse-phase high performance liquid chromatography. To avoid partial loss of methionine and cystine, samples of ingredients, diets, and feces were oxidized with performic acid to methionine sulfone and cysteic acid prior to acid hydrolysis. The apparent dry matter and protein digestive utilization coefficients varied from 68% to 109% and from 70% to 103%, respectively. Apparent digestibility of protein for casein, soy paste, wheat meal and wheat gluten were very high (over 90%), corn gluten and poultry byproducts meal showed high protein digestibility (over 80%), but porcine byproducts meal and sorghum meal had low digestibility (76% and 70%, respectively). There was a reasonable, but not total, correspondence between apparent protein digestibility and average essential amino acid digestibility coefficients, except for arginine in corn gluten, phenylalanine and leucine in sorghum meal, phenylalanine in soy paste and lysine in wheat meal and poultry by-product meal. The most digestible feed ingredients for whiteleg shrimp were: wheat gluten, wheat meal and soy paste; poultry byproduct meal and corn gluten were less digestible and the lowest digestibility occurred in porcine byproduct meal and sorghum meal. Feedstuffs exhibited great variability in dry matter, protein and amino acid digestive utilization coefficients, which should be considered when formulating shrimp feeds.