Bioefficacy of isolated compound l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester from entomopathogenic actinobacteria Actinokineospora fastidiosa against agricultural insect pests, human vector mosquitoes, and antioxidant activities.

Spodoptera litura and Helicoverpa armigera are polyphagous pests of agricultural crops in the Asian tropics since these pests have been responsible for massive crop and carry economic losses and low commodity production. At the same time, mosquitoes are vectors for numerous dreadful diseases, which is the most important group of insect for their public health concern. Using synthetic insecticides to control the pests can lead to contamination of land surface and groundwater and impact beneficial soil organisms and nontarget species. Applications of bioactive compounds are received considerable attention across the world as alternatives to synthetic insecticides. In the current study, actinobacterial secondary metabolite was isolated from Actinokineospora fastidiosa for the first time. The effect of actinobacterial metabolite (l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester) was assessed on agricultural pest S. litura and H. armigera, mosquito vectors larvae Ae. aegypti, An. stephensi, and Cx. quinquefasciatus. The bioactive fraction was characterized through UV, FTIR, and NMR analysis. GC-MS analyses reveal the existence of a bioactive compound with a respective retention time of 19.740 responsible for larvicidal activity. The bioefficacy of the l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester showed high antifeedant activity on S. litura (80.80%) and H. armigera (84.49%); and larvicidal activity on S. litura (82.77%) and H. armigera (88.00%) at 25 µg/mL concentration, respectively. The effective LC50 values were 8.07 µg/mL (F = 2.487, r2 = 0.988, P ≤ 0.05) on S. litura and 7.53 µg/mL (F = 123.25, r2 = 0.951, P ≤ 0.05) on H. armigera. The mosquito larvicidal effect of isolated compounds l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester treated against Ae. aegypti, An. stephensi, and Cx. quinquefasciatus the obtained percentage mortality was 96.66, 83.24, 64.52, 50.00, and 40.00% against Ae. aegypti; 100.00, 86.22, 73.81, 65.37, and 56.24% against An. stephensi; 100.00, 90.00, 76.24, 68.75, and 56.23% against Cx. quinquefasciatus. The mosquito larvae of Ae. aegypti obtained LC50 value was 13.25 µg/mL, F = 28.50, r2 = 0.90; on An. stephensi was 10.19 µg/mL, F = 15.55, r2 = 0.83, and Cx. quinquefasciatus was 9.68 µg/mL, F = 20.00, r2 = 0.87. Furthermore, l-isoleucine-, N-allyloxycarbonyl-, and dodecyl ester-treated larvae produced significant pupicidal activity on S. litura (62.71%) and H. armigera (66.50%) at 25 µg/mL, along with increased larval and pupal duration as compared to control group. Treated larvae revealed obliteration in the midgut epithelial cells and destruction of microvilli was noticed as compared to the control. The isolated compounds l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester did not produce any significant mortality on zebrafish embryos in all tested concentrations on biosafety observation. The potential microbial isolated molecule may fit well in IPM programs. Since the risk to human health, the environment, etc. is unknown.

Difference in the nutritional, in vitro, and functional characteristics of protein and fat isolates of two Indian chia (Salvia hispanica L) seed genotypes with variation in seed coat color.

This study aimed to determine the association between the seed coat color of two chia seed genotypes for their composition, protein content, amino acid, and fatty acid profiles. The optimal pH for protein isolation for both genotypes (BCPI and WCPI) was 10, based on protein purity and solubility. Fatty acid profiling indicated, overall, 18 different fatty acids higher in BCPI10 with linolenic acid domination (∼66%) followed by linoleic acid (∼19%) and oleic acid (∼6%), contributing PUFAs (∼86%). Optimized protein isolates, black (BCPI10) and white (WCPI10) chia, had shown purity, L*-value, solubility, and yields of 90.65%, 75.86%, 77.75%, 11.30%, and 90.00%, 77.83%, 76.07%, 10.69%, respectively. BCPI10 depicted higher EAA (33.19 g/100 g N) and EEA indices (57.676%) compared to WCPI10 (32.14 g/100 g N) and 56.360%, respectively. Amino acid profiling indicated higher, PER, TAA, TEAA, TNEAA, TAAA, TBA, acidic AA values for BCPI10, and higher leucine/isoleucine ratio for WCPI10 having leucine and sulfur amino acids as limiting amino acids. BCPI10 had higher sulfur-containing amino acid contents, as the main contributor to the albumin a water-soluble fraction, leading to its higher in vitro digestibility (71.97%) than WCPI10 (67.70%). Both isolates exhibited good WHC and OHC of 3.18, 2.39 and 3.00, 2.20, respectively. Both protein isolates had similar ∆Td (°C) values with some variation in FTIR spectrum from 1000 cm-1 to 1651 cm-1 having more peak intensity for BCPI10. SDS-PAGE indicated bands at 150 kDa, representing globulin and mild bands at 25-33 kDa for glutelin and albumin. A significant (p < 0.05) variation reported in this study for protein and lipid profiles of both genotype attributes to genetic differences between the seeds. PRACTICAL APPLICATION: Based on the nutritional profile, both chia seed isolates (black and white) are suitable for consumption with an edge for black seed when supplemented with their limiting amino acids. The high values of the functional properties and structural characteristics combined with high nutritional values make the chia protein isolate an excellent source of raw material for various food formulations. Fatty acid profile of the oils from the genotypes showed the presence of high amounts of unsaturated fatty acids, especially the PUFAs with more number of fatty acids in black chia seed. The excellent lipid profile of chia seed oil indicates the benefit of using chia seed oil as a source of essential fatty acids in the human diet for optimal health.

Ultra-HPLC-MS pseudo-targeted metabolomic profiling reveals metabolites and associated metabolic pathway alterations in Asian plum (Prunus salicina) fruits in response to gummosis disease.

Plum (Prunus spp.) is an economically and nutritionally important stone fruit that is grown worldwide. Gummosis disease (GD) is one of the most common limiting factors that adversely affects the yield and quality of stone fruits such as plum. Elucidating plum fruit metabolomics responses is essential to develop sustainable agricultural practices to combat GD in the future. Herein, an ultra-high-performance liquid chromatography coupled to mass-spectrometry (UHPLC-MS) pseudo-targeted metabolomic profiling was first performed to elucidate the overall metabolic alterations in Asian plum (Prunus salicina Lindl.) fruit in response to GD. The most pivotal differential metabolites, including certain amino acids and proanthocyanidins, in GD and control groups were identified by combining multivariate data analysis with strict statistical criteria. Metabolic pathway enrichment analysis showed that GD induced a series of coordinated defence responses and reprogramming of various metabolic pathways, including glucosinolate biosynthesis, 2-oxocarboxylic acid metabolism, valine, leucine and isoleucine degradation, and isoquinoline alkaloid biosynthesis pathways. Using UHPLC-MS-based pseudo-targeted metabolomic profiling, we systematically evaluated overall metabolic modifications in Asian plum fruits in response to GD for the first time. The identified metabolic pathway alterations helped to better understand the internal relationships and related metabolic networks.

PycnoRacer®, a fitness drink including Pycnogenol®, improves recovery and training in the Cooper test.

BACKGROUND: This study evaluates the effects of training (on running distance measured with a Cooper test) in 3 weeks in non-professional athletes using PycnoRacer®, a fitness drink (FD) including Pycnogenol® during the training period. METHODS: Pycnogenol® has been used in preclinical conditions and prevention. PycnoRacer® is a liquid combination of Pycnogenol® (15 mg), L-leucine (0.6 g), L-arginine (0.3 g), L-isoleucine (0.3 g), and L-valine (0.3 g). Two comparable groups (one using the FD) were formed: 60 training athletes were requested to drink one bottle 4 times/day, while 65 controls did not use PycnoRacer® or other comparable sports drinks. All subjects had a strong athletic background and advanced knowledge of the procedures of the tests. Two daily training sessions were completed. The sessions consisted in warming up and running a Cooper test. RESULTS: 125 subjects completed the three weeks according to plans. There were nine dropouts due to logistical or working problems. Subjects using the FD improved on average by 18.83% (range 12-23%) in their running distance with training. The difference with controls was significant (P<0.05) at 3 weeks (controls improved on average by 8.9%; range 3-17.4%). The difference between the two groups was on average 9.93% (P<0.05). A comparable increase in VO2max was observed in the groups. In the FD group the increase was on average by 10.05 mL/kg/min compared to 4.95 mL/kg/min in controls, with a difference of 5.1 mL/kg/min (P<0.05). A VAS score showed comparable values. Lower values (concerning muscular pain and cramps) were observed in FD subjects (P<0.05) at the end of the 3 weeks of training. The level of plasma free radicals (PFR) values after the last Cooper test was significantly lower at 3 weeks in the FD group (P<0.05). No intolerance problem was observed by subjects using the FD. CONCLUSIONS: In conclusion, the use of PycnoRacer® improved training, running distance, VO2max and PFR decreasing muscular pain and cramps.

Response of piglets to the standardized ileal digestible isoleucine, histidine and leucine supply in cereal-soybean meal-based diets.

Improving the amino acid (AA) profile of the diet by using l-Lys, l-Thr, dl-Met, l-Trp and l-Val helps to reduce the dietary CP content, thereby reducing nitrogen excretion while maintaining the performance of pigs. Valine is the fifth limiting AA in cereal-soybean meal-based diets. The extent to which the CP content in the diet can be reduced further without compromising performance depends on the requirement of the next limiting AA. In cereal-soybean meal-based diets, Ile, His and Leu may be the limiting AAs after Val, although information on the requirements for these AAs is scarce. Six experiments were conducted to determine the effect of supplementing a low-CP diet with l-Ile, l-His and l-Leu on the performance of pigs weighing 10 to 20 kg. Experiment 1 was designed to determine the most limiting AA with respect to performance among Ile, His and Leu. A diet 10% deficient in Ile, Leu and His relative to the National Research Council (NRC, 1998) requirement estimates tended to decrease daily feed intake and daily gain by 6% and 8%, respectively. A 10% deficiency in His alone had no effect, whereas a 10% deficiency in Ile or Leu slightly reduced daily feed intake and gain. In the remaining experiments, the standardized ileal digestible (SID) Ile : Lys, His : Lys and Leu : Lys requirements were estimated. In Experiments 2, 3, 4, 5 and 6, 14 blocks of six pigs each were assigned to six levels of SID Ile : Lys (40%, 43%, 46%, 49%, 52% and 55%), His : Lys (20%, 24%, 28%, 32%, 36% and 40%), His : Lys (21%, 24%, 27%, 30%, 33% and 36%), Leu : Lys (70%, 78%, 86%, 94%, 102% and 110%) and Leu : Lys (80%, 90%, 100%, 110%, 120% and 130%), respectively. Across experiments, the estimated SID Ile : Lys, His : Lys and Leu : Lys requirements for maximizing daily gain were 49%, 32% and 102%, respectively, using a curvilinear plateau model. When Ile, His and Leu levels were 10% below the requirement estimate, daily gain was reduced by 9%, 3% and 3%, respectively. The results of this study indicate that the Ile requirement estimate is lower than the current NRC requirement estimate, whereas the Leu and His requirements correspond to those proposed by the NRC.

Oncosecretomics coupled to bioenergetics identifies α-amino adipic acid, isoleucine and GABA as potential biomarkers of cancer: Differential expression of c-Myc, Oct1 and KLF4 coordinates metabolic changes.

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.

Determination of the fourth and fifth limiting amino acids in broilers fed on diets containing maize, soybean meal and poultry by-product meal from 28 to 42 d of age.

1. Valine (Val) is considered the 4th limiting amino acid for broilers fed on diets containing ingredients from vegetable origin. However, Val and Isoleucine (Ile) may be co-limiting for broilers fed on diets containing animal protein meals. An experiment was conducted to examine growth responses and meat yield of broilers provided diets varying in digestible Val (dVal) and digestible Ile (dIle) concentrations from 28 to 42 d of age. 2. Eight experimental diets varying in dVal (dVal to dLys ratios from 0·66 to 0·76) and dIle (dIle to dLys ratios from 0·57 to 0·67) were studied; digestible Lysine (Lys) was formulated to 9·9 g/kg in all diets. 3. Broilers fed on a negative control (NC) diet supplemented with crystalline Val (L-Val) and crystalline Ile (L-Ile), dVal to dLys = 0·76 and dIle to dLys = 0·67, grew faster and had higher breast meat yield than birds fed on NC + L-Ile (dVal to dLys = 0·66 and dIle to dLys = 0·67), NC + L-Val (dVal to dLys = 0·76 and dIle to dLys = 0·57), and NC + reduced L-Val and L-Ile (dVal to dLys = 0·71 and dIle to dLys = 0·62). 4. Feeding broilers on the NC + L-Val and L-Ile (dVal to dLys = 0·76 and dIle to dLys = 0·67) diets gave similar BW gain, carcase weight and yield and total breast meat weight and yield to birds fed on the positive control-fed broilers with no added L-Val and L-Ile (dVal to dLys = 0·76 and dIle to dLys = 0·67). 5. These results indicate that Val and Ile are co-limiting in diets containing poultry by-product meal.

Biochemical monitoring in fenugreek to develop functional food and medicinal plant variants.

Many plants used as functional foods or for medicinal purposes have been criticized for their inconsistent physiological effects. Variation in genotype and environmental conditions under which plants are produced can contribute to this inconsistency in biochemical composition. Fenugreek (Trigonella foenum-graecum L.) is a medicinal plant that not only can lower blood glucose and cholesterol levels in animals, but also can be used as a forage crop for livestock feed. Seed content for the bioactive compounds diosgenin, galactomannan and 4-hydroxyisoleucine was characterized for ten fenugreek genotypes under rainfed and irrigated conditions. High and low seed yielding genotype/environment combinations were identified that possessed distinct biochemical and seed production traits. In general high seed yielding genotype/environment combinations exhibited a more stable biochemical composition and consisted largely of irrigated fenugreek. This research indicates that comprehensive biochemical analysis of plant products would facilitate the development of more reliable produce for use by the functional food/medicinal plant industry.

The standardized ileal digestible isoleucine-to-lysine requirement ratio may be less than fifty percent in eleven- to twenty-three-kilogram piglets.

Most studies concerning the Ile requirement in pigs have been carried out using blood products as a protein source, and these have a relatively low Ile content relative to the other branched-chain AA (BCAA). There are indications that an excess supply of one BCAA can affect the utilization of the other BCAA. Little information is available concerning the Ile requirement in pigs when the supply of the other BCAA is moderate (e.g., in cereal- and soybean meal-based diets). The objective of the present study was to evaluate the response of piglets to Ile supplementation under different nutritional conditions. In all experiments, piglets were housed individually and had ad libitum access to feed during a 3-wk period. The first experiment was carried out to study the response of piglets to an increasing Ile supply by using 2 sources of l-Ile differing in degree of purity. Piglets received either a control diet with 48% standardized ileal digestible (SID) Ile:Lys or 1 of 4 other diets containing graded levels of either source of l-Ile to provide 52 or 56% SID Ile:Lys. All diets were formulated to provide 1.00% SID Lys in the diet. Feed intake and growth were not affected by Ile level or Ile source. Experiment 2 was performed to exclude a possible interaction between Ile and Lys supply. In a 2 x 2 factorial arrangement, 2 levels of Lys (1.00 and 1.15% SID Lys) and 2 levels of Ile (48 and 60% SID Ile:Lys) were used. Growth and G:F were 8 and 7% greater in piglets receiving the diet with the greater Lys content, but the Ile:Lys did not affect performance. No interactions were observed between the Lys and Ile supplies. In Exp. 3, a 2 x 2 factorial arrangement was used to test the effect of protein source (spray-dried blood cells or corn gluten meal) and Ile supply (50 or 65% SID Ile:Lys) on performance in piglets. Both protein sources had an elevated BCAA content but differed in Leu and Val contents. Protein source or Ile supply did not affect feed intake, growth, or G:F in the piglets. Plasma concentrations after an overnight fast reflected the difference in AA concentrations of the diets. In conclusion, the results of these experiments indicate that the SID Ile:Lys requirement may be not greater than 50% in piglets receiving cereal- and soybean meal-based diets with a moderate BCAA content. In contrast to other studies, we could not confirm that the Ile requirement was affected by BCAA content of the diet.

Response to lysine intake in composition of body weight gain and efficiency of lysine utilization of growing male chickens from two genotypes.

Male chickens of a broiler (B) and a layer (L) genotype were grown in floor pens from d 8 to 21 posthatch in groups of 10. Three pens per genotype were allocated to each of 10 experimental diets. The diets were offered ad libitum and they differed in lysine concentration from 3.8 to 16.8 g/kg. The source of supplemental lysine was L-lysine x HCl. All birds were killed at the end of the experiment, and representative birds (3 groups of 10 per genotype) were killed at the start for baseline measurements. Accretions of protein, fat, energy, and amino acids were determined by comparative body analysis. Responses were described with sigmoidal and exponential functions. Additionally, the net disappearance rate (NDR) of amino acids from the small intestine was studied with the basal diet (3.8 g of lysine/kg) using 6 replicated pens of 15 birds per genotype. Titanium dioxide was the indigestible marker. Net disappearance rates were not significantly different between genotypes for CP or any amino acid. Responses to incremental lysine concentration were nonlinear for both genotypes but distinctly different in magnitude between genotypes. Estimated y(max) values for 14-d BW, protein gain, and gain/ feed ratio were 534 (B) and 153 (L) g, 87.1 (B) and 28.7 (L) g, and 0.82 (B) and 0.71 (L) g/g. Protein accretion approached 95% of the estimated y(max) with dietary lysine concentrations of 12.5 (B) and 10.4 (L) g/kg. The amino acid profile of accreted whole body protein was different between genotypes, and was affected by supplementary lysine. Lysine content in accreted whole body protein approached upper values of 7.4 (B) and 5.6 (L) g/16 g of N with increasing dietary lysine concentration. Marginal efficiency of lysine utilization, determined as delta lysine accretion/delta lysine intake, showed maxima of 99% (B) and 74% (L). These maxima were achieved at intakes which were much lower than those needed for high protein accretion. It was concluded that the efficiency of amino acid utilization may depend on genotype, perhaps due to differences in the relative proportion of different protein fractions to whole body protein and due to differences in the ratio of synthesis and degradation of body proteins. Nonlinear relationships and different amino acid pattern of accreted body protein should be implemented in future models of requirements.

The pattern of intracellular free amino acids in granulocytes from hemodialysis patients change to an oral protein supplement (granulocyte amino acids after protein in HD patients).

MATERIALS AND METHODS: The concentrations of free intracellular amino acids in granulocytes and plasma amino acids, normalized protein nitrogen appearance rate, serum insulin-like growth factors, plasma proteins, anthropometric and bioimpedance measurements were determined before and after an oral protein supplement in 19 stable patients on maintenance hemodialysis in a randomized, double-blind placebo-controlled study with crossover after 3 months. The hemodialysis patients were well-nourished with an ideal body weight of 91% after both protein supplementation and after placebo. RESULTS: After protein supplementation (7.8 g/d) the intracellular concentration of valine, isoleucine, threonine and tyrosine and the valine/glycine and tyrosine/phenylalanine ratios in the cells were significantly increased (p < 0.05). In contrast, the concentrations of plasma amino acids, serum insulin-like growth factors, and plasma proteins and body weight and anthropometric and bioimpedance measurements were unchanged. Dialysis efficiency was unchanged throughout the study. CONCLUSIONS: The present study supports the conclusion that protein supplementation to well-nourished hemodialysis patients does not improve the nutritional status measured by plasma proteins, body weight, anthropometric and bioimpedance measurements. The increase in intracellular amino acid concentrations indicates better cellular nutrition and metabolic control.

Composition of ruminal bacteria harvested from steers as influenced by dietary forage level and fat supplementation.

The objective of this study was to examine the effects of dietary forage level and fat supplementation on the chemical composition of mixed ruminal bacteria (MRB). Six ruminally cannulated beef steers (354 kg +/- 18) were given ad libitum access to six diets (13.2% CP; DM basis) that were offered twice daily in a 6 x 6 Latin square design. Treatments were arranged as a 2 x 3 factorial with two forage levels (70 vs 30% of dietary DM as corn silage) and three forms of fat supplementation including no canola seed or canola seed added at 10% of dietary DM as whole treated with alkaline hydrogen peroxide or untreated crushed. Canola seed contributed 5% added fat to the total diet. The remaining dietary ingredients were corn, canola meal, molasses, and urea. No interactions (P > .05) between dietary forage level and canola seed supplementation were observed. Concentrations of OM, N, and all amino acids were higher (P < .05) in MRB from steers fed low forage than in MRB from steers fed high forage. Concentrations of purines and GE and the N:purines ratio in MRB were not affected (P > .05) by dietary forage level or canola seed supplementation. Canola seed supplementation did not affect (P > .05) concentrations of OM, N, or most of the amino acids in MRB. Concentrations of four essential amino acids (i.e., isoleucine, leucine, lysine, and phenylalanine) in MRB were decreased (P < .05) due to canola seed supplementation. Dietary forage level did not affect (P > .05) concentrations of long-chain fatty acids in MRB.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the lipids of Butyrivibrio fibrisolvens.

Butyrivibrio fibrisolvens strain D-1 was grown on a lipid-free chemically defined medium. The lipids were extracted with chloroform-methanol and separated into nonpolar and polar fractions by silicic acid column chromatography. Further separations were made by preparative thin-layer chromatography. The lipid fractions were identified by specific staining reactions and R(F) values, by phosphorus and nitrogen determinations, by chromatography of hydrolysis products, and by the use of infrared spectroscopy. The major nonpolar lipid was free fatty acid. Four major polar lipids were identified: phosphatidylethanolamine, phosphatidyl glycerol, lipoaminoacid, and glycolipid. The lipoaminoacid contained alanine, leucine, and isoleucine. The glycolipid contained galactose. The major fatty acids identified were C16:0 and C18:1. The significance of the presence of lipoaminoacid is discussed.