Prediction of apparent protein digestibility of ingredients and diets by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for juvenile Pacific white shrimp Litopenaeus vannamei

Aquafeed production faces global issues related to availability of feed ingredients. Feed manufacturers require greater flexibility in order to develop nutritional and cost-effective formulations that take into account nutrient content and availability of ingredients. The search for appropriate ingredients requires detailed screening of their potential nutritional value and variability at the industrial level. In vitro digestion of feedstuffs by enzymes extracted from the target species has been correlated with apparent protein digestibility (APD) in fish and shrimp species. The present study verified the relationship between APD and in vitro degree of protein hydrolysis (DH) with Litopenaeus vannamei hepatopancreas enzymes in several different ingredients (n = 26): blood meals, casein, corn gluten meal, crab meal, distiller`s dried grains with solubles, feather meal, fish meals, gelatin, krill meals, poultry by-product meal, soybean meals, squid meals and wheat gluten. The relationship between APD and DH was further verified in diets formulated with these ingredients at 30% inclusion into a reference diet. APD was determined in vivo (30.1 +/- 0.5 degrees C, 32.2 +/- 0.4%.) with juvenile L vannamei (9 to 12 g) after placement of test ingredients into a reference diet (35 g kg(-1) CP: 8.03 g kg(-1) lipid; 2.01 kcal g(-1)) with chromic oxide as the inert marker. In vitro DH was assessed in ingredients and diets with standardized hepatopancreas enzymes extracted from pond-reared shrimp. The DH of ingredients was determined under different assay conditions to check for the most suitable in vitro protocol for APD prediction: different batches of enzyme extracts (HPf5 or HPf6), temperatures (25 or 30 degrees C) and enzyme activity (azocasein): crude protein ratios (4 U: 80 mg CP or 4 U: 40 mg CP). DH was not affected by ingredient proximate composition. APD was significantly correlated to DH in regressions considering either ingredients or diets. The relationships between APD and DH of the ingredients could be suitably adjusted to a Rational Function (y = (a + bx)/(1 + cx + dx2), n = 26. Best in vitro APD predictions were obtained at 25 degrees C, 4 U: 80 mg CP both for ingredients (R(2) = 0.86: P = 0.001) and test diets (R(2) = 0.96; P = 0.007). The regression model including all 26 ingredients generated higher prediction residuals (i.e., predicted APD - determined APD) for corn gluten meal, feather meal. poultry by-product meal and krill flour. The remaining test ingredients presented mean prediction residuals of 3.5 points. A model including only ingredients with APD>80% showed higher prediction precision (R(2) = 0.98: P = 0.000004; n = 20) with average residual of 1.8 points. Predictive models including only ingredients from the same origin (e.g., marine-based, R(2) = 0.98; P = 0.033) also displayed low residuals. Since in vitro techniques have been usually validated through regressions against in vivo APD, the DH predictive capacity may depend on the consistency of the in vivo methodology. Regressions between APD and DH suggested a close relationship between peptide bond breakage by hepatopancreas digestive proteases and the apparent nitrogen assimilation in shrimp, and this may be a useful tool to provide rapid nutritional information. (C) 2009 Elsevier B.V. All rights reserved.

Apparent digestibility of selected feed ingredients in diets formulated for the sub-adult mud crab, Scylla paramamosain, in Vietnam

The present study was conducted to explore the potential to incorporate local plant-based feed ingredients into diets formulated for the mud crab species, Scylla paramamosain, commonly exploited for aquaculture in South-east Asia. Four test ingredients (defatted soybean meal, rice bran, cassava meal and corn flour) were incorporated at 30% or 45% inclusion levels in a fishmeal-based reference diet and used in digestibility trials where apparent digestibility coefficients (ADCs) for experimental diets and test ingredients were determined. Generally, high ADC values were obtained using diets containing 30% soybean meal or rice bran. By contrast, the lowest ADC values were obtained for the diet containing 45% cassava meal [70.9% for dry matter (ADMD); 77.1% for crude protein (ACPD) and 80.2% for gross energy (AGED)]. Similar trends were observed when ADC ingredient (I) digestibilities were compared. Specifically, the highest ADCI values were obtained for soybean meal when used at a 30% inclusion level (87.6% ADMDI; 98.4% ACPDI and 95.6% AGEDI) while the lowest ADCI values were obtained using cassava meal at a 45% inclusion level (53.8% ADMDI; 60.2% ACPDI and 67.3% AGEDI). Based on the current findings, we propose that soybean meal and rice bran could be considered for incorporation into formulated diets for S. paramamosain.

Effect of selected feed meals and starches on diet digestibility in the mud crab, Scylla serrata

The present study examined the capacity of the mud crab, Scylla serrata to digest experimental diets that contained different animal and plant-based feed meals or different levels or types of starch. The apparent dry matter digestibility (ADMD) coefficients for all feed meals tested in the first part of this study, except meat meal, were similar (78–88%). Crude protein digestibility (ACPD) coefficients for all feed meals were relatively high, with values ranging from 86% to 96%. Cotton seed meal, poultry meal, canola meal, fishmeal, soybean meal and lupin meal had similar gross energy digestibility (AGED) values (P>0.05) ranging from 84% to 89%. In the second part of this study, the impact of selected starches on the digestibility of fishmeal-based formulated diets was assessed. The apparent starch digestibility (ASD) of wheat starch decreased significantly as the inclusion level was increased from 15% to 60%, however, there was no significant effect on ACPD values. At a 30% inclusion level, the ASD of diets containing different starches decreased in the order corn>wheat>potato=rice. Moreover, ACPD values were significantly higher (P<0.05) in the diets containing corn or rice starch than in those containing wheat or potato starches.

Using Alkanes to Predict Voluntary Intake, Faecal Output and Digestibility of Buffel Grass Hay Fed to Steers

The cuticular waxes of forage plants contain long chain n-alkanes with odd carbon chain lengths in the range C25-C37 which are quantitatively recovered in faeces. When these concentrations are used with the concentrations of administered synthetic even chain length alkanes, the voluntary intake (VI), faecal output (FO) and digestibility (DMD) of forages can be estimated (Dove and Mayes 1991, 1996).

Apparent digestibility of some nutrient sources by juvenile mud crab, Scylla serrata (Forskal 1775)

As part of preliminary work aimed at the development of a formulated diet for the mud crab, Scylla serrata, an experiment was conducted with juvenile mud crabs (95.65±2.17 g) to determine apparent digestibility coefficients (ADC) for cellulose, fish meal, shrimp meal, blood meal, soybean meal, wheat flour and cod liver oil. Apparent digestibility coefficients for dry matter (ADCdm), energy (ADCenergy) and protein (ADC protein) were in the ranges 70.0-95.7%, 77.4-97.1% and 57.7-97.9% respectively. Soybean meal had the highest ADCdm and wheat flour had the lowest value (P<0.05), while the ADCdm for fish meal, blood meal and shrimp meal were not different (P?0.05). Similarly, soybean meal had the same ADCenergy as that of fish meal, but higher than those of cod liver oil, blood meal and shrimp meal (P<0.05). Moreover, the ADC protein for blood meal or shrimp meal were not significantly different from fish meal (P?0.05); nevertheless, they were lower than that of soybean meal and higher than that of wheat flour (P<0.05). Of significant interest was the ADCdm (78.0%) and ADCenergy (77.4%) for cellulose, which indicates that plant-based nutrient sources may well be a useful component of formulated diets for mud crabs.

Differences between the in vitro digestibility of extrusa collected from oesophageal fistulated steers and the forage consumed.

In a study that included C-4 tropical grasses, C-3 temperate grasses and C-3 pasture legumes, in vitro dry matter digestibility of extrusa, measured as in vitro dry matter loss (IVDML) during incubation, compared with that of the forage consumed, was greater for grass extrusa but not for legume extrusa. The increase in digestibility was not caused by mastication or by the freezing of extrusa samples during storage but by the action of saliva. Comparable increases in IVDML were achieved merely by mixing bovine saliva with ground forage samples. Differences were greater than could be explained by increases due to completely digestible salivary DM. There was no significant difference between animals in relation to the saliva effect on IVDML and, except for some minor differences, similar saliva effects on IVDML were measured using either the pepsin-cellulase or rumen fluid-pepsin in vitro techniques. For both C-4 and C-3 grasses the magnitude of the differences were inversely related to IVDML of the feed and there was little or no difference between extrusa and feed at high digestibilities (>70%) whereas differences of more than 10 percentage units were measured on low quality grass forages. The data did not suggest that the extrusa or saliva effect on digestibility was different for C-3 grasses than for C-4 grasses but data on C-3 grasses were limited to few species and to high digestibility samples. For legume forages there was no saliva effect when the pepsin-cellulase method was used but there was a small but significant positive effect using the rumen fluid-pepsin method. It was concluded that when samples of extrusa are analysed using in vitro techniques, predicted in vivo digestibility of the feed consumed will often be overestimated, especially for low quality grass diets. The implications of overestimating in vivo digestibility and suggestions for overcoming such errors are discussed.

Enhancing digestibility of pastures by cattle using kangaroo bacteria

Enhancing digestibility of native pastures by cattle using kangaroo fibrolytic bacteria.

Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

Destruction Of Rat-Liver Microsomal Cytochrome-P-450 Invitro By A Monoterpene Ketone, Pulegone A Hepatotoxin

Significant destruction (68%) of liver microsomal cytochrome P-450 and homogeneous cytochrome P-450 purified from PB-treated rats is noticed upon incubation with 10 mM pulegone at 37-degrees-C for 30 min. There is also a concomitant loss of heme. The destructive phenomenon does not require metabolic activation of pulegone. The destruction of purified cytochrome P-450 is time-dependent and saturable. Structure-activity studies suggest that an alpha-isopropylidine ketone unit with a methyl positioned para to the isopropylidine group as in pulegone is necessary for the in vitro destruction of cytochrome P-450. SKF-525A at a concentration of 4-mM obliterates the destruction of cytochrome P-450 by pulegone. Experiments with C-14-pulegone suggest that pulegone or its rearranged product binds covalently to the prosthetic heme of cytochrome P-450.

Studies on the macroconida of microsporum-canis-characteristics of invitro amino-acid incorporativing system

The characteristics of an in vitro polyuridylic acid dependent amino acid incorporating system prepared from germinating macroconidia of Microsporum canis are described. The incorporation of 14C-phenylalanine into polyphenylalanine is dependent on S-30 extract, adenosine triphosphate, magnesium ions and polyuridylic acid. Incorporation is slightly enhanced by yeast transfer ribonucleic acid and pyruvate kinase. The system is highly sensitive to ribonuclease, puromycin and miconazole (an antifungal agent), moderately sensitive to sodium fluoride and much less sensitive to phenethylalcohol, cycloheximide, chloramphenicol and deoxyribonuclease. Cell-free extract from ungerminated conidia has less capacity to synthesize the protein and during germination a marked increase in the protein synthetic activity is observed. The results from experiments wherein ribosomes and S-100 fraction from germinated and ungerminated spores are interchanged, revealed that the defect in the extract from the ungerminated spore is in the ribosomes.