Evaluation of protein hydrolysis in raw sources by digestive proteases of Senegalese sole (Solea senegalensis,Kaup 1858) using a combination of an in vitro assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of products

The hydrolysis of protein in different animal and plant sources by the intestinal proteases of Senegalese sole (Solea senegalensis) was studied using a combination of an in vitro digestibility assay and the evaluation of the protein fractionation by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). In vitro hydrolysis was performed for 90 min in a closed reactor maintained at constant pH and temperature. Samples of the reaction mixture at different time intervals were developed in SDS‐PAGE gels to evaluate the progressive hydrolysis of the different protein fractions in each protein source. A numerical value [coefficient of protein degradation (CPD)], which integrates the information obtained after image analysis of the gels, is proposed for comparison among proteins, according to the intensity of the hydrolysis produced by sole proteases. Additionally, the total free amino acid released from proteins was measured during the in vitro assay. Casein, squid meal and soybean concentrate (SBC) proteins showed very similar degradation patterns, with a quick and almost complete proteolysis within the first minutes of the enzymatic reaction. Fish and krill meals were hydrolysed more progressively. On the contrary, pea meal (PM) and corn gluten meal (CGM) showed scarce changes in their protein profile after 90 min of reaction. For animal protein sources, the final CPD values ranged from 77.6% to 87.0%, showing not significant differences. By contrast, PM (30.5%) and CGM (32.3%) presented significantly lower CPD values (P<0.05) as compared with SBC (90.6%). In general, a linear fitting was found between CPD and the release of free amino acids during in vitro protein hydrolysis. The present study provides detailed information, which, combined with the conventional in vitro digestibility studies, may help in the evaluation of different raw sources according to their protein degradation patterns. This information can be applied directly to estimate the protein nutritional quality of ingredients for Senegalese sole feeds.     

热塑挤压蒸煮对鱼肉蛋白质营养价值的影响初探

采用胃蛋白酶—胰酶复合处理法对原料鱼肉、蒸煮和罐藏处理的鱼肉产品,以及热塑挤压蒸煮的鱼肉制品进行蛋白质体外消化率的测定,分析比较各种鱼肉制品的蛋白质体外消化率的测试结果;结果表明,挤压蒸煮对鱼肉蛋白质的消化率有一定的影响。     

Amino acid retention of gilthead sea bream (Sparus aurata,L.) fed with pea protein concentrate

A trial of 218 days of duration was carried out to assess the use of pea protein concentrate (PPC) as a substitute for fish meal in diets for juvenile gilthead sea bream (52 g average initial weight), using four diets (0, 16, 32 and 48) with PPC inclusion levels of 0, 162, 325 and 487 g kg^?1, respectively. At the end of the trial, the fish reached weights of 397, 385, 383 and 355 g for 0, 16, 32 and 48 diets, respectively; diet 48 gave the lowest specific growth rate (SGR, 0.88% per day) but 0%, 16% and 32% PPC did not present statistical differences. Feed conversion ratio (FCR), protein efficiency ratio (PER) and body composition were not affected by the diets. Digestible protein retention was significantly higher with the 0% diet, but no energy retention differences were observed. Retention rates of ingested and digestible amino acids were very similar; in general, the retention of essential amino acids decreased as dietary PPC increased, and lysine retention was the highest and phenylalanine retention was the lowest. From the results of this experiment, it may be concluded that fish meal can be replaced up to 32% by PPC in sea bream without negative effects on fish weight gain, FCR and PER.     

不同脂肪源对黄颡鱼幼鱼生长性能、体成分、血清生化指标、体组织脂肪酸组成及抗氧化能力的影响

为探讨脂肪酸激活过氧化物酶体增殖物激活受体 (PPARα)在吉富罗非鱼脂代谢中的降脂效应,实验采用离体和在体试验相结合的方法进行研究。实验初期选用7种单体脂肪酸［油酸 (OA)、棕榈酸 (PA)、亚油酸 (LA)、α-亚麻酸 (ALA)、花生四烯酸 (AA)、二十碳五烯酸 (EPA)、二十二碳六烯酸 (DHA)］孵育罗非鱼原代肝细胞,并采用荧光定量PCR检测脂代谢相关指标。结果显示,与对照组相比,ALA、AA和EPA显著上调PPARα、肉碱棕榈酰基转移酶 (CPT1)、脂酰辅酶A氧化酶 (ACO)等脂肪酸分解代谢关键基因表达,ALA还分别上调脂肪水解脂肪甘油三脂脂肪酶 (ATGL)、激素敏感脂肪酶 (HSL)和脂肪酸转运相关基因脂蛋白酯酶 (LPL)的表达,EPA则同时抑制脂合成基因乙酰辅酶A羧化酶 (ACC)的表达,ALA、EPA和AA均能降低细胞内甘油三酯 (TG)含量。PA和OA则使细胞内TG含量显著上升。综合数据,筛选出2种不同基因激活模式效应脂肪酸EPA和ALA设计在体实验,分别设置正常脂 (7%)组、高脂 (12%)组以及高脂分别含ALA (0.6%、1.2%)、EPA (0.6%、1.2%)的6组半纯化饲料,饲喂初始体重为(3.53±0.03) g的吉富罗非鱼8周。结果显示,各组间生长无显著差异,与高脂组相比,4个处理组均能显著降低罗非鱼体脂,且随着ALA和EPA含量增加,肥满度、肝体脂数、肠系膜脂肪指数逐渐降低,血清和肝脏TG和胆固醇含量降低。血清谷草转氨酶、谷丙转氨酶、超氧化物歧化酶各组间无显著差异。EPA (1.2%)组肝脏丙二醛含量显著升高,还可显著提高肝脏脂代谢相关基因 (PPARα、CPT1、ACO、ATGL、HSL)的表达,促进脂分解代谢及降脂。研究表明,饲料含0.6%的EPA或ALA即可上调PPARα及其脂分解代谢相关靶基因表达,发挥降脂效应。本研究结果为饲料配制与脂肪源筛选提供参考。

     

Evaluation of an in vitro digestibility technique for the prediction of protein digestibility in the South African abalone, Haliotis midae L.

A pH-stat multienzyme in vitro digestibility assay was investigated for its efficacy in predicting apparent protein digestibility coefficients in abalone diets. Linear regression analysis between in vitro digestibility estimates and in vivo digestibility coefficients obtained from abalone, revealed that the technique could be used to reliably predict apparent protein digestibility. Maximal predictability of the system was obtained when protein sources were analysed according to origin – animal or plant. The technique was used to assess the apparent protein digestibility of 34 potential protein sources for use in formulated feeds for Haliotis midae .     

Apparent digestibility coefficients of selected feed ingredients for dourado Salminus brasiliensis

The excellence of its flesh and fast growth makes the dourado, Salminus brasiliensis , a carnivorous fish native to the Prata basin, a potential candidate for intensive fish farming. This study evaluated the apparent digestibility coefficients (ADC) of energy and nutrients of animal and plant protein sources for the carnivorous Characin dourado S. brasiliensis . Fish (19.5 ± 5.0 g) were stocked in plastic cages (80-L) and fed pelleted test diets containing 30% of the test ingredient [fish meal (FM), poultry by-product meal (PBM), soybean meal (SBM), and corn gluten meal (CGM) plus 70% of a reference diet (481.4 g kg⁻¹ of crude protein and 18.63 kJ of gross energy per gram]. After the last daily meal, cages were transferred to cylindrical, conical-bottomed aquarium (200-L) where faeces were collected by sedimentation in a refrigerated container. Except for ADC of protein and energy, all other ADC of nutrients showed significant differences ( P  < 0.01). ADC values were: 94.3%, 91.3%, 93.1%, and 93.5% for crude protein; 91.0%, 90.3%, 87.8%, and 88.8% for gross energy; 92.1%, 84.5%, 80.6%, and 79.3% for ash; 83.9%, 80.3%, 84.3%, and 84.6% for dry matter; 97.4%, 96.7%, 93.3%, and 91.5% for lipid for FM, PBM, SBM, and CGM, respectively. The average amino acid ADC was: 93.6%, 90.0%, 92.1%, and 92.5% of FM, PBM, SBM, and CGM, respectively. All test ingredients were efficiently used and can partially replace FM in diets for carnivorous dourado.     

Digestibility of nutrients and amino acids of different protein sources in practical diets by largemouth bass Micropterus salmoides (Lacepéde, 1802)

This study evaluated the apparent digestibility coefficients (ADCs) of dry matter, protein, lipid, energy, calcium, phosphorus, and essential and non‐essential amino acid availability of animal and plant protein sources in practical diets for the carnivorous fish, largemouth bass Micropterus salmoides (8.0±0.5 g; 10.0±0.3 cm). Fish were conditioned to accept artificial feed and those stocked in plastic cages were fed pelleted test diets. Diets contained 30% of the ingredient tested mixed with 70% of a reference ration (RD) containing 40 g 100 g^?1 of crude protein, 19.85 kJ g^?1 crude energy, and 0.5% of chromic oxide. Cages were then transferred to cylindrical, conical‐bottomed aquaria (200 L) where the faeces were collected by sedimentation in a refrigerated container. All the tested ingredients had high ADCs for protein and lipid; ADCs of amino acids of varying protein sources showed significant differences (P<0.01). Plant protein sources did not significantly influence the ADCs of diets. The results allow the inference that the availability of amino acids in plant and animal protein sources varies considerably.     

Protein quality of larval feed with increased concentration of hydrolysed protein: effects of heat treatment and leaching

Four heat coagulated early weaning diets with increasing concentrations of pepsin hydrolysed protein, were investigated with regard to the change in protein quality during feed production and exposure to leaching. Water-soluble N, trichloroacetic acid-soluble N and amino acid (AA) profiles were determined in finished diets and in diets leached for 6 min. In vitro diet digestibility was measured and related to increasing inclusion of hydrolysed protein and N leakage. Seventeen to 47% of soluble N in the feed ingredients was made insoluble by heat denaturation during feed production, but the concentration of peptides and free amino acids (FAA) were not influenced. All peptides/FAA and 70–80% of water-soluble protein were lost after exposure to leaching. Increased inclusion of hydrolysed protein increased the loss of crude protein (15–30%). All taurine and 30% of histidine was lost during leaching, no other major changes in AA profile were found. There was no difference in digestibility between diets exposed to leaching. However, leached diets showed reduced digestibility as compared to diets that had not been exposed to leaching. In conclusion micro-bound type diets as used in this study have a low efficiency in delivering soluble N to fish larvae and should be carefully considered for this purpose.     

Prediction of culture performance of juvenile Litopenaeus vannamei by in vitro (pH-stat) degree of feed protein hydrolysis with species-specific enzymes

Rapid in vitro methods for measuring digestibility may be useful in analysing aqua feeds if the extent and limits of their application are clearly defined. The pH‐stat protein digestibility routine with shrimp hepatopancreas enzymes was previously related to apparent protein digestibility with juvenile Litopenaeus vannamei fed diets containing different protein ingredients. The potential of the method to predict culture performance of shrimp fed six commercial feeds (T3, T4, T5, T6, T7 and T8) with 350 g kg^?1 declared crude‐protein content was assessed. The consistency of results obtained using hepatopancreas enzyme extracts from either pond or clear water‐raised shrimp was further verified in terms of reproducibility and possible diet history effects upon in vitro outputs. Shrimps were previously acclimated and then maintained over 56 days (initial mean weight 3.28 g) on each diet in 500‐L tanks at 114 ind m^?2, clear water closed system with continuous renewal and mechanical filtering (50 μm), with four replicates per treatment. Feeds were offered four times daily (six days a week) delivered in trays at feeding rates ranging from 4.0% to 7.0% of stocked shrimp biomass. Feed was accessible to shrimp 4 h daily for 1‐h feeding period after which uneaten feed was recovered. Growth and survival were determined every 14 days from a sample of 16 individuals per tank. Water quality was monitored daily (pH, temperature and salinity) and managed by water back flushing filter cleaning every 7–10 days. Feeds were analysed for crude protein, gross energy, amino acids and pepsin digestibility. In vitro pH‐stat degree of protein hydrolysis (DH%) was determined for each feed using hepatopancreas enzyme extracts from experimental (clear water) or pond‐raised shrimp. Feeds resulted in significant differences in shrimp performance (P < 0.05) as seen by the differences in growth rates (0.56–0.98 g week^?1), final weight and feed conversion ratio (FCR). Shrimp performance and in vitro DH% with pond‐raised shrimp enzymes showed significant correlation (P < 0.05) for yield (R² = 0.72), growth rates (R² = 0.72–0.80) and FCR (R² = ?0.67). Other feed attributes (protein : energy ratio, amino acids, true protein, non‐protein nitrogen contents and in vitro pepsin digestibility) showed none or limited correlation with shrimp culture performance. Additional correlations were found between growth rates and methionine (R² = 0.73), FCR and histidine (R² = ?0.60), and DH% and methionine or methionine+cystine feed contents (R² = 0.67–0.92). pH‐stat assays with shrimp enzymes generated reproducible DH% results with either pond (CV ≤ 6.5%) or clear water (CV ≤ 8.5%) hepatopancreas enzyme sources. Moreover, correlations between shrimp growth rates and feed DH% were significant regardless of the enzyme origin (pond or clear water‐raised shrimp) and showed consistent R² values. Results suggest the feasibility of using standardized hepatopancreas enzyme extracts for in vitro protein digestibility.     

A comparative study on the apparent digestibility of selected feedstuffs in hybrid catfish (Clarias macrocephalus × Clarias gariepinus) and Nile tilapia (Oreochromis niloticus)

The apparent digestibility (AD) of dry matter (DM), organic matter (OM), crude protein (CP) and essential amino acids (EAA) in five selected feedstuffs was evaluated in hybrid catfish and Nile tilapia. The AD of DM, OM and CP in cassava leaf meal (CLM) was lower (P < 0.05) than in groundnut meal (GNM), soybean meal (SBM), sesame husk meal (SSH) and shrimp head meal (SHM). The AD of DM, OM and CP among GNM, SBM, SSH and SHM was not different (P > 0.05). The AD of most EAA was higher in SSH (P < 0.05) than in CLM, GNM, SBM and SHM. The AD of most EAA among CLM, GNM, SBM and SHM was not different (P > 0.05). The AD of DM and OM in CLM was higher (P < 0.05) in hybrid catfish than in Nile tilapia, while there was no difference in the AD of CP in CLM between fish species. The AD of DM, OM, CP and EAA among GNM, SBM, SSH and SHM did not differ between hybrid catfish and Nile tilapia. In conclusion, there were only small differences in the nutritional properties between the selected feedstuffs in both fish species. Most EAA in the selected feedstuffs were equally well utilized by hybrid catfish and Nile tilapia.     

团头鲂对7种饲料的蛋白质、氨基酸及磷的表观消化率

本实验以0.5%的三氧化二铬(Cr2O3)为指示剂,研究分别以鱼粉、肉骨粉、豆粕、花生粕、棉粕、酒精糟及其残液干燥物(DDGS)和菜粕作为蛋白饲料,将这7种饲料原料与基础日粮按照3:7的比例组成实验日粮,观测团头鲂(Megalalobrama amblycephala Yih)对这7种饲料的干物质、粗蛋白、氨基酸及总磷的表观消化率.实验鱼初始体质量为(296.54±1.71)g,分别投喂相应的实验饲料5周后采用自排法收集粪便.结果表明,7种饲料原料的干物质、蛋白质、总氨基酸以及总磷的表观消化率的范围分别为53.65%～77.48%、78.66%～93.63%、80.12%～94.83%、3.21%～48.02%.其中粗蛋白及总氨基酸的表观消化率均以菜粕最高;花生粕、豆粕与鱼粉相接近;肉骨粉最低.磷的表观消化率以DDGS最高,菜粕和肉骨粉最低,其他4种原料的磷的表观消化率范围为11.81%～29.10%.由此可见,菜粕、豆粕、花生粕可作为团头鲂优质的植物蛋白源,在实际生产配方中可部分替代鱼粉.DDGS蛋白消化率高于肉骨粉,且磷的表观消化率较高,也可作为一种优质的鱼用饲料原料.     

Apparent digestibility coefficients of feed ingredients and their predictability in compound diets for gilthead seabream, Sparus aurata L.

Apparent digestibility of crude protein, amino acids, lipid, carbohydrate and energy was measured for a range of feed ingredients fed to gilthead seabream, Sparus aurata L. — fish meal, poultry meal, meat meal, blood meal, squid meal, extracted soyabean and wheat flour. Chromic oxide was used as a non-absorbed reference substance and faeces were collected by stripping. Diets compounded from mixtures of these ingredients were then used to examine the possibility of predicting the digestibility of formulated diets.
Apparent digestibility of crude protein ranged from 79% to 90%, lipids from 83% to 95% and energy from 72% to 88% in the different ingredients. Apparent digestibility of carbohydrates was lower and ranged from 49% to 77%. Apparent digestibility of amino acids was higher than that of crude protein and differences were found among digestibilities of individual amino acids.
Tests conducted using five compound diets indicated that ingredient digestibility was additive for protein, amino acids, lipids and energy, whereas the digestibility of carbohydrates in the compound feeds was slightly lower than predicted.
Diets for Sparus aurata may thus be formulated on the basis of digestibility of individual ingredients.     

草鱼对27种饲料原料中氨基酸的表观消化率

本试验以0.5％Cr2O3为指示物,按照“70％基础饲料 30％试验原料“的饲料配制方法,在可控条件的室内循环养殖系统中用2龄草鱼种（体重150-200g）对常用的饲料原料的氨基酸表观消化率进行测定。试验选用了进口鱼粉、国产鱼粉、蟹粉、肉粉、肠衣粉、酵母、菜饼、黄菜粕、菜粕、双低菜籽粕、豆粕、膨化大豆、芝麻饼、棉粕、玉米胚芽粕、玉米蛋白粉和酒糟粉共17种商品蛋白质饲料原料,玉米、麦麸、次粉、米糠、标粉、小麦、大麦、玉米糟、稻谷、药渣共10种能量饲料原料,得到了每种原料的16种氨基酸的表观消化率。表观消化率主要规律表现为：草鱼对原料蛋白质消化率高时对氨基酸的总消化率也高,如对鱼粉、豆粕的消化率高;对玉米类原料如玉米蛋白粉、玉米渣、玉米的氨基酸消化率较低,而对小麦类原料如小麦粉、麦麸、次粉等的消化率相对较高;在植物饼粕类中,草鱼对豆粕和菜粕的消化率较高,而对棉粕的消化率则相对较低;对同种原料的16种不同的氨基酸的表观消化率也有很大的差异,这种差异的规律性较差。