Enzymatic Hydrolysate Of Casein Research Articles

Determination of ileal endogenous nitrogen losses and true ileal nitrogen digestibility during non-steady-state conditions of the 15N-isotope dilution technique

ABSTRACT The aim was to determine ileal endogenous nitrogen losses (ENL) and true ileal N-digestibility (TD-N) under non-steady-state conditions of the 15N-isotope dilution technique (15N-IDT), using diets generating low and high ENL and compare results to those obtained under steady-state conditions. Twelve growing pigs (mean LW 22.4 kg) fitted with a post-valve T-caecum cannula were fed an enzyme-hydrolysed casein (EHC)-based diet or an EHC diet + 4% quebracho tannins (QT) and were labelled via continuous 15N-leucine i.v. infusion or twice daily oral 15N-leucine administration. Digesta were collected daily over three consecutive hours with blood plasma sampled on the four consecutive days after cessation of 15N-labelling. There was a significant effect of sampling day on the dilution factor. Endogenous N losses were significantly lower for the EHC than the EHC+QT diet (2.41 vs. 8.69 g/kg DMI), while no significant effect of sampling day was observed. The TD-N of the EHC+QT diet did not differ from the TD-N of the EHC diet (95.1 vs. 92.0%). A significant effect of sampling day was observed for TD-N with day 1 and 2, being higher than day 4. Non-steady-state conditions overestimated ENL by 25–28% as compared to 3 h collections in steady-state conditions, but the relative overestimation was similar for the EHC diet as for the EHC+QT diet. TD-N did not differ significantly compared to 12 h steady-state measurements, but comparison to 3 h steady-state measurements showed that non-steady-state conditions overestimated TD-N for the EHC+QT diet by 9%. However, on day 4 this overestimation disappeared. Using the 15N-IDT during non-steady-state conditions can provide valuable additional data on endogenous N losses and TD-N.

A Stable and Efficient Genetic Transformation Method for Embryogenic Callus of Fraxinus mandshurica

Fraxinus mandshurica is a widely used greening and ornamental tree species. However, its genetic transformation system has been hampered by problems such as low transformation efficiency, among others, which can hinder research related to molecular breeding and the analysis of functional genes. Thus, in this study, a novel genetic transformation method for efficient transformation of the embryonic callus of Fraxinus mandshurica was investigated. The method was optimized in terms of factors such as antibiotics, infection solution concentrations, co-culture time, and somatic embryo maturation. The results indicated that the optimal antibiotic concentration was 10 mg·L−1 of hygromycin (Hyg). At this point, the callus proliferation multiple was only 0.12. The highest transformation efficiency was found to be 93.93% when the absorbance of the infection solution concentration at OD600 was 0.4. Interestingly, transformation efficiency was found to be highest (77.9%) at 48 h of co-culture, with a GUS staining rate of 88.23%. The medium for somatic embryo maturation of transformed callus was half-strength MS medium (MS 1/2) containing 60 g·L−1 polyethylene glycol, 1 mg·L−1 abscisic acid, 400 mg·L−1 casein enzymatic hydrolysate (CH), 20 g·L−1 sucrose, 1 g·L−1 activated charcoal, and 5 g·L−1 gellan gum. The medium for somatic embryo germination was MS ½, containing 0.2 mg·L−1 of N-(Phenylmethyl)-9H-purin-6-amine(6-BA) and 5.0 mg·L−1 of gibberellin (GA). These results are of significance for the verification of the gene function and future genetic improvement of Fraxinus mandshurica.

Purification and identification of xanthine oxidase inhibitory peptides from enzymatic hydrolysate of α-lactalbumin and bovine colostrum casein

The development of food-derived Xanthine Oxidase (XO) inhibitors is critical to the treatment of hyperuricemia and oxidative stress-related disease. Few studies report on milk protein hydrolysates’ XO inhibitory activity, with the mechanism of their interaction remaining elusive. Here, different commercial enzymes were used to hydrolyze α-lactalbumin and bovine colostrum casein. The two proteins hydrolyzed by alkaline protease exhibited the most potent XO inhibitory activity (bovine casein: IC50 = 0.13 mg mL−1; α-lactalbumin: IC50 = 0.28 mg mL−1). Eight potential XO inhibitory peptides including VYPFPGPI, GPVRGPFPIIV, VYPFPGPIPN, VYPFPGPIHN, QLKRFSFRSFIWR, LVYPFPGPIHN, AVFPSIVGR, and GFININSLR (IC50 of 4.67–8.02 mM) were purified and identified from alkaline protease hydrolysates by using gel filtration, LC-MS/MS and PeptideRanker. The most important role of inhibiting activity of peptides is linked to hydrophobic interactions and hydrogen bonding based on the results of molecular docking and molecular dynamics simulation. The enzymatic hydrolysate of α-lactalbumin and bovine colostrum casein could be a competitive candidates for hyperuricemia-resisting functional food.

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry.

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography–tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.

First Report of Chaetomium globosum Causing Leaf Blight on Brassica oleracea L. in China.

Cabbage (Brassica oleracea var. capitata L.) is widely cultivated in China and be of important economic value. In October 2019, all the plants of cabbage inbred line '2358' cultivated in greenhouses of the Chinese Academy of Agricultural Sciences (Beijing) were showing symptoms of leaf wilt. It usually took two weeks for the leaves to get completed wilted and the plants gradually died. It was approximately 550 plants affected, and 30 plants were collected and processed as samples. Symptomatic leaves were cut into small pieces (5×5 mm), surface sterilized with 75% ethanol for 30 s, and then sterilized with 8% NaClO for 3 mins, rinsed three times in sterile distilled water, plated on complete medium (CM: 3g Casein Enzymatic Hydrolysate + 3g Casein Acid Hydrolysate + 6g Yeast Extract+10g Sucrose + 15g Agar + 1L dH2O) and incubated at 27℃ for 6 days. Subsequently, the purified culture was obtained by tissue isolation and single-spored on CM. The colony on CM was up to about 50mm and 70mm in diameter after 4 and 7 days, respectively (Supplementary Fig. 2). At the same time, the edge of the colony gradually turned brown. Microscopic observation found that the diameter of the mycelium was about 5 µm (Supplementary Fig. 3). The conidia were transparent white and ovoid-shaped, about 0.3-0.5 µm in diameter (Supplementary Fig. 4). Fungus in liquid CM were spherical with surface hairs (Supplementary Fig. 1), which was consistent with the morphological characteristics of Chaetomium globosum. The rDNA internal transcribed spacer (ITS) of isolate ZM2019 was amplified using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'- TCCTCCGCTTATTGATATGC-3') (Hong et al. 2013). The amplified product was sequenced and deposited in GenBank. The 573-bp amplicon (GenBank accession no. MN833403) showed a 100% homology to C. globosum isolate CES5 (MN173145). According to the morphological and molecular characterization, the fungus causing leaf blight on '2358' was identified as C. globosum. In order to further distinguish the pathogen species, the β-tubulin (tub2) gene region were amplified using primers T-F (5'-CCCCTGAACTACCCCACC-3') and T-R (5'-TATTTGACCCGACTGACC-3') and sequenced. Finally, we further identified this pathogen as C. globosum CBS 148.51 (Wang et al. 2016), according to the blast result of the sequence (GenBank accession no. MW252170) in National Center for Biotechnology Information (NCBI). To confirm the pathogenicity of this fungus, the leaves of 12 healthy seedlings of '2358' were inoculated by spraying 106 conidia/ml suspension and 12 seedlings inoculated with sterile water served as controls. All plants were incubated in a growth chamber maintained at 27℃. Ten days after inoculation, the leaves of all plants became wilted, which were consistent with the symptom in the natural state, while the control plants remained disease-free (Supplementary Fig. 6b and Fig. 6c). Subsequently, C. globosum was isolated from the inoculated seedlings again. The entire process from isolation to inoculation was repeated again and the same results were obtained. Re-isolation of C. globosum and inoculation of the host fulfiled Koch's postulates. C. globosum has been reported previously to occur on many horticulture plants such as Punica granatum (Guo et al. 2015) and Cannabis sativa (Chaffin et al. 2020); but no brassica species has been reported so far as susceptible to C. globosum. In this sense, this is the first report of leaf blight caused by C. globosum on cabbage in China, in greenhouse condition.

Intensified pollination and fertilization ameliorate heat injury in rice (Oryza sativa L.) during the flowering stage

Heat induces spikelet sterility during flowering in rice. Spikelet sterility in heat-stressed plants is associated with poor pollination due to insufficient pollen grain deposition and unsatisfactory fertilization from reduced pollen germination on the stigma. Measures aimed at facilitating pollen shedding and pollen germination will help to address the adverse effects of heat stress on spikelet fertility in rice during the flowering stage. Intensified pollination by artificially assisted pollen shedding combined with intensified fertilization by promoting pollen germination through chemical application (pH=8.0, 10 % sucrose, 0.1 mM spermidine, 0.01 % myo-inositol, 0.01 % boric acid, 1 μM 24-epibrassinolide, 1 mM KCl, 1 mM CaNO3, 1 mM CaCl2, 0.03 % casein enzymatic hydrolysate, 0.01 % ferric ammonium citrate, 10 mM γ-aminobutyric acid, and 1–2 drops of Tween 20) increased spikelet fertility significantly compared with that without intensified measures under heat treatments in controlled greenhouse conditions. Intensified pollination increased the percentage of anther dehiscence and the number of pollen grains on the stigma, and the chemical application increased the number of germinated pollen grains on the stigma, thus increasing the spikelet fertility and ultimately the grain yield. In open-field conditions, intensified pollination and fertilization measures increased the spikelet fertility, grain weight and quality significantly under elevated temperatures. The proposed methods of enhancing pollination and fertilization are simple and effective for ameliorating the adverse effects of heat stress on the grain yield and quality in rice. This study provides strategies for sustainable rice production under the high temperatures expected with future global warming.

Usage of nutrient Medium Based on Dry Hydrolysate of Casein in Manufacturing Bivalent Chemical Cholera Vaccine

Objective of the study was to select the standardized substrate containing dry hydrolysate of casein for preparation of nutrient medium utilized for manufacturing bivalent chemical cholera vaccine under submerged cultivation of cholera vibrio strains in fermenters. Materials and methods . We used Vibrio cholerae O1 strains of classical biovar: strain 569B Inaba and strain M-41 Ogawa. Examined were two dry substrates of the medium: enzymatic hydrolysate of casein, Type I Himedia (India) and pancreatic hydrolysate of casein, produced by the State Scientific Center of Applied Microbiology and Biotechnology (Russian Federation). Produced under laboratory conditions at the premises of the RusRAPI “Microbe” medium was used as a control. Submerged cultivation was conducted in bioreactors during (9±1) h with aeration and automatic feeding of glucose and ammonia. Production of protective antigens was measured applying immunochemical and biological methods. Results and discussion . It is demonstrated that submerged cultivation of cholera vibrio production strains on nutrient media under study provides for synthesis of protective antigens the parameters of which comply with the requirements of normative documentation. More standardized and higher indicator values of the target product are ensured by cultivation of producer strains on nutrient medium with a substrate from dry enzymatic hydrolysate of casein, containing (1.5±0.1) g/l of amino nitrogen for the strain V. cholerae M-41 and (2±0.1) g/l – for V. cholerae 569 B. Transition to the use of standardized dry protein components of cultivation media does not lower the quality of the chemical cholera vaccine, but allows for the reduction of cost price and duration of technological process.

Bioactive hydrolysates from casein: generation, identification, and in silico toxicity and allergenicity prediction of peptides.

Bioactive casein peptides have attracted considerable attention for their applications in industry. However, there is little clarity regarding mass spectrometric profiles for peptides in enzymatic hydrolysates of casein produced under varying conditions. In this study, the compositions of the peptides from casein hydrolysates were compared for different enzyme/substrate ratio (E/S) and hydrolysis times. The toxicity, allergenicity and bioactivity of the identified peptides were assessed in silico. A total of 70 unique peptides were identified, and there were 28, 21, 13 and 8 peptides from αs1 -casein, αs2 -casein, β-casein and κ-casein respectively. The peptide number decreased with the increase in E/S and hydrolysis time. Moreover, peptides with relative molecular mass Mr ranging from 1000 to 1500 Da occupied the highest proportion of 31.43%, and almost all of the peptides showed Mr less than 5000 Da. In silico analysis showed that all of the peptides were non-toxic and non-allergenic, and several of them were assessed by PeptideRanker as having a relatively high likelihood of being bioactive peptides. Composition of the peptides in the casein hydrolysates varied with the enzymolysis conditions. This study's results may facilitate the production of target bioactive peptides by controlling E/S and hydrolysis time, which is beneficial for the application of casein peptides in the functional food industry. © 2017 Society of Chemical Industry.

Direct plant regeneration from in vitro-derived shoot apical meristems of finger millet (Eleusine coracana (L.) Gaertn.)

A rapid and reproducible protocol for direct plant regeneration has been established using in vitro-derived, actively growing shoot apical meristems (SAMs) of the genotype 'CO(Ra)-14'. Shoot apical meristems from 3-day-old seedlings of three finger millet genotypes viz, 'CO(Ra)-14', 'Try-1', and 'Paiyur-2' were assessed for the efficiency of direct shoot induction. The regeneration efficiency of 'CO(Ra)-14' surpassed the other two genotypes and produced a mass of green, multiple shoots within 12 d on Murashige and Skoog medium containing 17.6 μM 6-benzylaminopurine (BA), 0.9 μM 2,4-dichlorophenoxyacetic acid in combination with 750 mg L−1 proline, 500 mg L−1 casein enzymatic hydrolysate, and 2 mg L−1 glycine. 'CO(Ra)-14' showed a maximum frequency of multiple shoot regeneration (96%) with an average of 8.3 shoots per explant. The age and size of shoot apical meristems played a crucial role in triggering adventitious shoot bud proliferation. The multiple shoot buds elongated spontaneously upon reducing the concentration of BA (to 11.0 μM) in the shoot induction medium. The in vitro proliferated shoots were rooted best in half-strength MS medium containing 2.8 μM indole-3-acetic acid and successfully acclimated in the field, subsequently developing into fertile plants. Thus, we report a short tenure (45 d) in vitro whole plant regeneration system, which could be effectively utilized for the production of transgenic finger millet plants.

Optimization of expression of untagged and histidine-tagged human recombinant thrombin precursors in Escherichia coli.

The present study is focused on preparation of proper Escherichia coli expression system to ensure high yields of various modified precursors of human recombinant thrombin, a potential biopharmaceutical reagent. Two thrombin precursors, the smallest single-chain α-thrombin precursor prethrombin-2 and its shortened form prethrombin-2∆13, and their His-tagged forms were used. In order to determine the effect of the different lengths and amino acid compositions of affinity His-tag on the target protein expression level, a variety of the His-tag sequences were used. We found out that the protein expression efficiency was closely related to the codons used for encoding of amino acids of fusion histidine tag. Optimization of culture medium composition is another way to increase yield of the target protein. Suitable medium composition can ensure cell growth to high densities which is related to total yield of expressed protein. In this study, a new optimized complex medium for batch fermentation was developed. Addition of nutrients like a yeast extract and enzymatic casein hydrolysate to the defined medium components had a positive impact on protein expression, where relatively high expression level of the target protein from total amount of cellular proteins was achieved. Further, we have focused on trace element solution composition, and the optimized nickel and selenium concentrations were determined. Our results show that the composition of essential trace metal solution has a major impact not only on expression level, but it can also affect cell growth rate.

Endogenous proteins in the ileal digesta of adult humans given casein-, enzyme-hydrolyzed casein- or crystalline amino-acid-based diets in an acute feeding study

To ascertain if the form of dietary nitrogen (free amino acids (AA), small peptides, or intact protein) affects the endogenous nitrogen containing substances lost from the upper digestive tract of humans. Digesta were collected via a naso-ileal tube from the terminal ileum of 16 adult humans in a single parallel study following an acute feeding regimen. Subjects were given an iso-nitrogenous and isocaloric test meal containing 150 g of casein (CAS) (n=6), enzyme-hydrolyzed casein (HCAS) (n=5) or crystalline AA (n=5) dissolved in 550 ml of water, as the sole sources of nitrogen. The mean concentrations and flows of total nitrogen, protein nitrogen, and soluble protein nitrogen passing the terminal ileum were significantly higher (P <0.01) for the CAS and HCAS test-meal groups compared to the AA meal group. Dietary CAS and HCAS had a considerable influence on digesta mucin concentrations and flows compared to free AA (+41%). Only 3-4% of the total nitrogen remained unidentified. The form of dietary nitrogen (protein, small peptides or free AA) had an acute effect upon the secretion or reabsorption of endogenous proteins in the small intestine of healthy humans, as evident from significant differences in both the quantity and composition of the proteins found in digesta at the end of the ileum.

Nutrient digestibility of lentil and regular- and low-oligosaccharide, micronized full-fat soybean fed to grower pigs1

A study was conducted to determine the standardized ileal digestibility (SID) of AA and calculate the NE value for regular-oligosaccharide, micronized full-fat soybean (R-MFFSB), low-oligosaccharide, micronized full-fat soybean (LO-MFFSB), lentil, and enzymatically hydrolyzed casein (EHC) for growing pigs. Six ileal-cannulated barrows (31.4 kg BW) were fed 6 diets in a 6 × 6 Latin square. Five diets were cornstarch based, containing either soybean meal (SBM), R-MFFSB, LO-MFFSB, or EHC as sole protein source or N free. The sixth diet contained lentil as sole protein and energy source. The SID of AA for diets was calculated using the N-free diet. Digestibility of AA in feedstuffs was determined by the direct method. Energy digestibility in SBM, R-MFFSB, and LO-MFFSB was determined by difference from the N-free diet whereas energy digestibility in lentil was determined by the direct method. On DM basis, SBM, R-MFFSB, LO-MFFSB, and lentil contained 52, 43, 43, and 27% CP, 8, 12, 14, and 16% NDF, and 1.8, 19, 21, and 1.6% ether extract, respectively. The SID of Lys for SBM was greater (P < 0.05) than that for R-MFFSB or LO-MFFSB (76 vs. 79 and 79%). The SID of other indispensable AA (except Trp) for SBM was also greater (P < 0.05) than that for R-MFFSB or LO-MFFSB. The R-MFFSB and LO-MFFSB were similar in SID of AA. The SID of Lys for lentil (81%) was lower (P < 0.05) than that for SBM with a similar trend for SID of other indispensable AA except for Met and Thr whose SID was similar to SBM. The SID of AA for EHC ranged from 98 to 112%. The SBM had a lower (P < 0.05) NE value than R-MFFSB or LO-MFFSB (2.63 vs. 2.95 and 3.00 Mcal/kg DM). Lentil and SBM were similar in NE value (2.60 vs. 2.63 Mcal/kg DM). In conclusion, R-MFFSB and LO-MFFSB were similar in energy and AA value for pigs. Lentil had lower SID of AA than SBM. However, lentil and SBM were similar in NE value; therefore, lentil can serve as alternative pulse feedstuff for pigs. The AA in EHC were mostly completely digested indicating that EHC can be fed to estimate ileal endogenous AA losses.

Nutritional Studies on Production of Antibacterial Activity by the Zebra Mussel Antagonist, Pseudomonas fluorescens CL0145A

Pseudomonas fluorescens strain CL0145A was discovered at the New York State Museum Field Research Laboratory as an effective agent against the environmentally destructive zebra mussel, which has contaminated US waters. Dried cells of the microbe are being commercialized as an environmentally friendly solution to the problem. We found that antibiotic activity against the Gram-positive bacterium Bacillus subtilis is produced and excreted by this strain. We have carried out studies to optimize production of the antibiotic. Studies were begun in a complex corn meal medium. Activity was found in both cells and culture supernates and was maximal after one day of fermentation. Static fermentation conditions were found to be superior to shaken culture. Production of extracellular antibiotic in complex medium was found to be dependent on the content of sucrose and enzymehydrolyzed casein. Indeed, production was greater in sucrose plus enzyme-hydrolyzed casein than in the complex medium. Of a large number of carbon sources studied as improvements over sucrose, the best was glycerol. An examination of nitrogen sources showed that production was improved by replacement of enzymehydrolyzed casein with soy hydrolysates. Production in the simple glycerol-Hy-Soy medium was not improved by addition of an inorganic salt mixture or by complex nitrogen sources, with the exception of malt extract. In an attempt to keep the medium more defined, we studied the effect of amino acids and vitamins as replacements for malt extract. Of 21 amino acids and 7 vitamins, we found tryptophan, glutamine, biotin, and riboflavin to be stimulatory. The final medium contained glycerol, Hy- Soy, tryptophan, glutamine, biotin, and riboflavin.

Prediction of apparent, standardized, and true ileal digestible total and reactive lysine contents in heat-damaged soybean meal samples1

Forty-two individually housed entire male pigs weighing 37.5 ± 0.15 kg (mean ± SEM) were used in a randomized block design having 7 dietary treatments (n = 6). The dietary treatments were 5 semisynthetic diets containing 350 g/kg of soybean (Glycine max) meal (SBM) with variable heat treatments (0, 7, 14, 21, and 28 min autoclaved at 135°C), a protein free (N-free) diet, and an enzymatically hydrolyzed casein (EHC) diet. Heat treatment linearly decreased (P < 0.001) total Lys content from 27.5 to 19.2 g/kg and reactive Lys content from 23.4 to 11.7 g/kg. Apparent, standardized, and true ileal digestible total and reactive Lys contents linearly decreased (P < 0.001) with increasing severity of heat treatment and were accurately predictable (P < 0.001) from total and reactive Lys content in heat-damaged SBM. These data indicate that excessive heat processing of SBM reduced both the content (P < 0.001) and digestibility (P < 0.001) of total and reactive Lys in SBM.

Increased dietary sodium chloride concentrations reduce endogenous amino acid flow and influence the physiological response to the ingestion of phytic acid by broiler chickens

1. A total of 240 Ross 308 broilers were used to investigate the effect of sodium (1·5 or 2·5 g/kg), phytate-P (0 or 3·2 g/kg), and phytase (0 or 1000 FTU/kg; 2x2x2 factorial) on endogenous amino acid flow using the enzyme-hydrolysed casein method. 2. The ingestion of phytate increased endogenous amino acid flow (∼30%) compared with the phytate-free control diets. Phytase reduced endogenous amino acid flow only when fed in concert with phytate, resulting in a significant phytate x phytase interaction. 3. Increasing dietary sodium concentration from 1·5 to 2·5 g/kg reduced endogenous amino acid flow by around 10%. This reduction of endogenous flow was particularly evident in diets which contained phytate, resulting in a significant sodium x phytate interaction for several amino acids, including Thr and Ser. Further, high sodium concentrations muted the effect of phytase resulting in a significant sodium x phytase interaction for some amino acids. 4. The concentration of Asp, Thr, Ser and some other amino acids was increased in the endogenous protein in response to the ingestion of phytate. Both sodium and phytase essentially restored the composition of endogenous protein to that of the phytate-free control. Further, as both sodium and phytase had similar effects there were significant interactions between sodium and phytase for most amino acids, such that one was only effective in the absence of the other. 5. These data confirm previous reports that phytate is a nutritional aggressor, causing quantitative and qualitative changes in endogenous protein flow. However, this is the first report which has shown that dietary sodium concentrations play a role in the severity of this antinutritional effect and consequently may blunt the efficacy of exogenous phytase. The mechanism is obscure, though it has been previously demonstrated that sodium can disrupt phytate:protein complexes, thus mitigating one of the mechanisms by which phytate exerts its antinutritional effect.

3H-leucine single-injection method for determining endogenous amino acid losses of broilers

Objective A 3H-leucine ( 3H-Leu) single-injection method was proposed for determining the endogenous amino acid losses of broilers. This method was based on the hypothesis that the ratio of the specific radioactivity (SR) of endogenous Leu in excreta (SR e) to that of free Leu in trichloroacetic acid–soluble plasma (SR p) remains constant after a single subcutaneous injection of 3H-Leu into birds fed different diets. Methods Two experiments were designed to clarify this hypothesis. In experiment 1, 40 female broilers were randomly divided into four groups and were force-fed a nitrogen-free diet (NFD), NFD plus enzyme-hydrolyzed casein (EHC), 5% crude protein (CP) and SBM (soybean meal), or 20% CP-SBM. In experiment 2, 24 broilers were randomly divided into four groups and were fasted or force-fed the NFD, 20% CP-SBM, or 20% CP and cottonseed meal (CSM) diet. After the forced feeding, broilers were administered 3H-Leu by a single subcutaneous injection at a rate of 30 μCi/kg of body weight. Blood samples were taken at 5 min, 30 min, 4 h, 24 h, 36 h, and 48 h after the injection. The excreta were totally collected and pooled over the 48-h experiment. Results The ratios of SR e to SR p remained the same for the birds force-fed the NFD, NFD + EHC, and 5% CP-SBM diets in experiment 1 and for the birds fasted and force-fed the NFD diet in experiment 2. The proportions of endogenous Leu to total Leu in excreta were 72.8%, 61.4%, and 57.5% for birds force-fed with the 20% CP-SBM diet in experiment 1 and 20% CP-SBM and 20% CP-CSM diets in experiment 2, respectively. Broilers fed the 5% CP-SBM and 20% CP-SBM diets excreted more ( P < 0.05) endogenous Leu than those fed the NFD and NFD + EHC diets in experiment 1. Broilers fed the 20% CP-SBM diet excreted more ( P < 0.05) endogenous Leu than those fed the NFD diet and fasted and the 20% CP-CSM diet was intermediate ( P > 0.05) in experiment 2. Conclusion The present study verified the hypothesis that the ratio of SR e to SR p remains constant after a single subcutaneous injection of 3H-Leu into broilers and proposes a new method to determine endogenous amino acid losses of broilers.

ORIGINAL ARTICLE: Molecular size distribution affects portal absorption rate of casein amino acids in rats

The aim of the study was to determine if differences in the molecular size of two protein sources affect in vivo intestinal absorption rates of amino acids under normal feeding conditions. Accordingly, the portal absorption rate of amino acids was studied in rats fed semi-synthetic diets containing native casein (NC) or enzymatically hydrolysed casein as the only protein sources. Enzymatic casein hydrolysate (ECH) consisted of a mixture of free amino acids (51.2% with respect to total amino acids) and low molecular weight peptides. Rats were pre-adapted to the experimental diets for 5 days prior to the absorption studies. Total free amino acid concentrations in portal vein plasma of rats fed ECH diet at 60, 105, 150 and 195 min after feeding were lower (p < 0.05) than those of rats fed NC. Lower (p < 0.05) concentrations of free threonine, proline, tyrosine, valine and tryptophan at all time points, and higher (p < 0.05) leucine at 60 and 105 min were found in rats fed ECH when compared with those fed NC in portal vein plasma. Portal flow rates of threonine, proline, tyrosine and valine were higher in NC at most time points tested, while leucine and lysine were higher for ECH fed rats 60 and 105 min after feeding. In arterial plasma, significantly (p < 0.05) higher concentrations of some individual free amino acids (proline, tyrosine, valine and tryptophan) were determined at 60, 105, 150 and 195 min after feeding, and lower leucine values after 60 and 105 min, in rats fed NC compared with those fed ECH. Results indicate that in normal feeding conditions amino acids from NC and ECH are absorbed at different rates in rats.

Debittering of protein hydrolysates using immobilized chicken intestinal mucosa

Enzymatic hydrolysates of casein and soybean were treated with alginate immobilized chicken intestinal mucosa, as an aminopeptidase source, to bring about debittering. The mucosa was hygienised by irradiation (20 kGy) which brought about a complete decontamination of the tissue accompanied by a 20% loss in aminopepidase activity. The effectiveness of the process was demonstrable by a higher acceptability and a marked reduction in bitterness scores for casein (from 4.4 to 2.5) and soybean (from 3.8 to 2.2) in organoleptic analysis. The action of aminopeptidases to bring about this change was corroborated by a concomitant increase in free amino acids and a decrease in average peptide length of the samples after treatment. The RP HPLC profiles of casein and soybean protein hydrolysates before and after treatment showed a higher content of peaks in the hydrophilic region suggesting a decrease in hydrophobic peptides, responsible for bitter taste, in both the samples. Immobilization of the mucosal tissue in alginate afforded an increased pH and temperature tolerance to the enzymes. The possibility of the system for continuous operation over extended time periods is also discussed.

Influence of Dietary Phytic Acid and Source of Microbial Phytase on Ileal Endogenous Amino Acid Flows in Broiler Chickens

The effects of phytic acid and 2 sources of exogenous phytase (bacterial vs. fungal) on the flow of endogenous amino acids at the terminal ileum of broilers were assessed using the enzyme-hydrolyzed casein method. Phytic acid (as the sodium salt) was included in a purified diet at 8.5 and 14.5 g/kg, and each diet was fed without or with a fungal (Aspergillus niger-derived) or a bacterial (Escherichia coli-derived) microbial phytase at 500 phytase units/kg of diet. Increasing the concentration of phytic acid in the diet from 8.5 to 14.5 g/kg increased (P < 0.001) the flow of all measured amino acids by an average of 68%, with a range from 17% for proline to 145% for phenylalanine. The flow of endogenous aspartic acid, serine, glutamic acid, glycine, leucine, tyrosine, phenylalanine, and histidine were increased by more than the mean, indicating changes in the composition of endogenous protein in response to the presence of higher concentrations of phytic acid. Supplementation of both phytases reduced (P < 0.001) the flow of endogenous amino acids, but the reduction (P = 0.06) was greater for the bacterial phytase compared with the fungal phytase. These data suggest that a substantial part of the amino acid and energy responses observed following phytase supplementation in broiler chickens stems from reduced endogenous amino acid flows and that the capacity of different phytases to counteract the antinutritive properties of phytic acid vary.

Aggregation properties of aqueous casein hydrolysate solutions at different pH

Fluorescence spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), nuclear magnetic resonance (NMR), and differential scanning calorimetry (DSC) were used to investigate the aggregation behaviour of casein hydrolysates at three pHs (2.0, 7.0 and 10.0). A three-dimensional macroaggregate network was formed via the association of globular aggregates at pH 2.0 and 7.0. However, the degree of association of globular aggregates was low at pH 10.0. The nanostructure was more compact at pH 2.0 and looser at pH 10.0. Furthermore, studies with a pyrene probe indicated that casein enzymatic hydrolysate (CEH) aggregates may represent viable vehicles to sequester hydrophobic, insoluble organic molecules.