Livestock Research for Rural Development 29 (3) 2017 Guide for preparation of papers LRRD Newsletter

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Effects of inclusion of protein hydrolysis from Tra catfish by-product waste water in the diets on apparent ileal digestibility and total tract retention coefficients of local chickens

Nguyen Thi Thuy and Nguyen Cong Ha1

Department of Animal Sciences, College of Agriculture and Applied Biology, CanTho University,Campus II, 3/2 Street, Ninh Kieu District, CanTho City, Viet Nam.
1 Department of Food Technology, College of Agriculture and Applied Biology, CanTho University,Campus II, 3/2 Street, Ninh Kieu District, CanTho City, Viet Nam.


An experiment was conducted to determine apparent ileal digestibility and total tract retention of diets with inclusion of Tra catfish by-product protein hydrolysate by enzyme (HWWE) or by organic acid (HWWO), or with fish meal (FM) and soya bean meal (SB) suplementation in the diets for Noi local chickens. The digestibility was conducted with 24 male Noi chickens at 8 weeks of age and were placed in individual pens to collect excreta and ileal digesta. Four treatments HWWED, HWWOD, FMD and SBD were introduced to determine the apparent ileal digestibility (AID) and total tract retention (ATD) of nutrients using Chromic oxide (Cr2O3) as indigestible marker. After 4 days of colleting excreta, the chickens were killed to collect the ileal digesta.

Results showed that, the AID and ATD of crude protein (CP) and ether extract (EE) were higher in HWWED and HWWOD diets compared to that of FMD or SBD diets. There were significantly increase total amino acid digestibility especialy isoleucine, leucine and lysine; and ileal digestibility of isoleucine, leucine, lysine, methionine and threonine of catfish by-product protein hydrolysate than fish meal and soya bean meal in the diets.

Key words: amino acid, catfish by-products, hydrolysate, ileal digestibility


Tra catfish (Pangasius hypophthalmus) occupy 95% freshwater catfish production in Vietnam, and processing by-products amount to about 65% of weight of the whole fish, which are input materials for catfish by-product meal processing, these by-products includes the head, bone, skin and scrap meat that remain after the two side fillets have been ground fresh, boiled, and the oil removed, then dried to produce catfish by-product meal. In this process, large amounts of processing waste water are discarded especialy in small scale factories, where have no modern machine system to use this in the process, and release to environment outside, lead to a wasted resource that cause enviromental polution.

Research from Dang Minh Hien et al (2015) were hydrolyzed this kind of by-product such as head, bone and scrap meat to produce powder protein hydrolysate product and fed to pigs (Nguyen Thi Thuy and Nguyen Cong Ha (2016). So the aim of this study was to hydrolysis of protein products from this processing waste water through enzymatic methods or organic acids to determine the appropriate level of hydrolysis of each method. And assessing the effectiveness of using hydrolyzed protein preparations in the diets of chikens, and determining digestibility evaluation. In order to do that, the project used enzyme and chemical methods to hydrolyze protein from Tra catfish processing waste water to short protein, peptide and amino acid that easy to absorbed by the chickens. In addition, using effectively these waste water sourse not only can reduce pollution of environment in small scale producers but also would decrease the cost of chicken production. But so far no study has been carried out on the effect of hydrolysate waste water on the digestibility of nutrients in chickens.

Therefore, the main objective of this research is to determine digestibility of hydrolysate waste water to compare with the others protein sources such as fish meal or soya bean meal.The expected results from the research will contribute to the advance poultry nutrition as well as with reduce feed cost and environmetal polution.

Materials and methods

Animal and experimental design

The experiment was conducted at the housholder in Binh Thuy district, Can Tho city. 24 Noi chickens at 8 weeks of age were placed into individual metabolic cages and distributed into 6 group of 4 treatments. The metabolic cages were made with 30 cm x 30 cm x 50 cm in width, length and height, respectively. The trial will be designed as completely randomized design with 4 treatments and 6 replications. Birds were weighed at the beginning and the end of the trial and at the beginning and end of excreta collection.

Experimental diets

Experimental diets were formulated with 60% of diet CP from energy feed ingredients (basal ingredients), which including rice bran, broken rice and maize meal. And 40% from protein feed sources, which are including fish meal (FM) , soy bean meal (SB), Tra catfish hydrolysate waste water (powder form) with enzyme (HWWE), or with organic acid (HWWO) supplementation in the diets.

Table 1. Ingredients and chemical composition (*) of the experimental diets(**)

Ingredients, %






Broken rice





Rice bran





Maize meal





Fish meal


Soya bean meal











Chemical composition of diets ,% of DM


























ME (MJ/kg) calculation





(*) DM: Dry matter; CP: Crude protein; EE: Ether extract; OM: Organic matter
(**) FMD: Basal ingredient (B) + fishmeal; SBD: B + Soya bean meal; HWWED: B + catfish by-product waste water hydrolyzed with enzyme; HWWOD: B + catfish by-product waste water hydrolyzed with organic acid.

The experimental diets were :

FMD : Basal ingredients + Fish meal

SBD : Basal ingredients + Soya bean meal

HWWED : Basal ingredients + HWWE

HWWOD : Basal ingredients + HWWO

Feed and supplementation

The production of HWWE and HWWO was carried out as described by Dang Minh Hien et al (2015). Briefly, catfish by-product waste water was collected from the small scale catfish by-product meal factories as a soluble matter, which was then centrifuged and extracted to get a protein solution, then was concentrated to 10 % (DM), the optimum concentration for protein hydrolysis by the enzyme bromelain (HWWE) or with organic acid as lactic acid (HWWO). Hydrolysis was carried out by adjusting the pH to 6.5 and temperature to 55oC, found to be the optimal conditions for hydrolysis. The enzyme bromelain (or lactic acid) was added to the protein solution at a the ratio of 1.5 mg bromelain/1,042 g protein for 120 minutes (similar with lactic acid). The protein hydrolyzate solution was finally transported to a dryer with the previous solid matter and dried to produce the final products, then these preparation products were fed to chickens in digestibility trial.

Procedure and measurement

The total tract retention (ATD) of OM, CP, EE, Ash and amino acids of experimental diets were measured by total excreta collection. And apparent ileal digestibility (AID) coefficients of amino acids were calculated using Cr2O3 as an indigestible marker.The trial were recorded 15 days, in which total feed intake was recorded daily individually. After 1 week of adaptation period, excreta was collected daily for 4 days from plates under the cages and stored frozen (-20 oC) prior to analysis. Then excreta collection were followed by a further 3-day adaptation period, 24 h fasting and 4 h free access to feed after which the birds were slaughtered for the collection of ileal digesta (Perttila et al 2002). This procedure were conducted as quickly as possible in order to minimise changes in digesta composition, feeding and killing were started at the same time for the whole group. Digesta were collected from the terminal ileum by gently flushing with distilled water into plastic containers. The ileum is defined as that portion of the small intestine extending from the vitelline diverticulum to a point 60mm proximal to the ileo-caecal junction (Huang et al 2005). Digesta samples of individual birds were pooled and stored at -20oC before chemical analysis.


The ATD is express in terms of the difference between the intake and that excreta as faeces as a proportion of amount consumed (McNab 1994).

As fomular:

Apparent ileal digestibility coefficients of amino acids were calculated using Cr2O3 as an indigestible marker:

Chemical analysis

The chemical composition of feed diets, excreta and digesta were determined using the following Association of Official Analytical Chemists methods (AOAC 1990). Dry matter (DM) was measured by drying the fresh samples at 105oC until dry. Crude protein was determined by the Kjeldahl method. Total ash was the residue after ashing the samples at 550 oC and organic matter (OM) was calculated by difference. The ether extract (EE) was determined by Soxhlet extraction. Amino acid concentration of the feed, excreta and ileal digesta were analyzed using HPLC (Spackman et al 1958). Chromium was measured by atomic absorption spectrophotometer after ashing and digesting the sample in a mixture containing perchloric and nitric acid (Fenton and Fenton 1979).

Statistical analysis

Data collected was analyzed by ANOVA using the General Liner Model (GLM) of Minitab Statistical Software Version 16. Tukey pair-wise comparisons were used to determine differences between treatment means at P<0.05.

Results and discussions

Apparent total and ileal digestibility of dietary nutrients

Table 2. Apparent ileal (AID) and total tract retention (ATD) (*) of the experimental diets(*)







Total tract retention,%





























Ileal digestibility, %





























(*) CP: Crude protein; EE: Ether extract; OM: Organic matter
(**) FMD: Basal ingredient (B) + fishmeal; SBD: B + Soya bean meal; HWWED: B + catfish by-product waste water hydrolyzed with enzyme; HWWOD: B + catfish by-product waste water hydrolyzed with organic acid.

The AID of OM, CP, EE and Ash in all diets were lower than that in the ATD, it because there is extensive bacterial activity in the caeca of chickens, stimulating effect of the gut micro-flora on protein synthesis. In addition, the fermentation occurs in the caeca of poultry influence the nutrient contents of excreta and thus modify results for digestibility (Short et al 1999). So, many researchers focused on digestibility trials in poultry at ileal level rather than excreta one because ileal digestibility can give accurate digestibility data due to eliminating hind gut micro flora effects (Applegate et al 2004).

Also the diets with HWWE or HWWO supplementation showed higher in both AID and ATD than that from FMD or SBD. It may be the waste water used to produce the HWWE or HWWO mostly constituted head, bone, scrap meat and skin after boiling to produce catfish by-product meal, thus making the CP content in the waste water higher than that in the fish meal or soya bean meal. Moreover, the HWWE or HWWO was produced from catfish head, bone, scrap meat and skin with controlled proteolytic enzyme digestion which hydrolyzed the protein into peptides and free amino acids (Folador et al 2006), and a high-quality animal protein source that provides an adequate supply of essential amino acids and fatty acids (Thuy et al 2007).

Recently, some studies have been conducted on the utilization of catfish by-products to produce protein hydrolysate using alcalase enzyme (Minh 2014) or using bromelain enzyme (Dang Minh Hien et al 2015). These studies showed that most of the protein in Tra catfish by-products undergoes complete hydrolysis to some low molecular weight peptides and high concentrations of amino acid mixtures. Therefore, the CP digestibility values showed significant difference among diets. Especialy, the values were lower in FMD and SBD diets compared to HWWED or HWWOD diets. In fact, Minh (2014) found that protein hydrolysate can be used to improve or modify the physicochemical and functional properties, such as solubility, fat absorption or sensory properties of proteins without loss of nutritional value. Theoretically, Protein hydrolysate consists mainly of low molecular weight peptides which can be absorbed rapidly and have higher nutrient digestibility. Furthermore, the explanation for the positive effects of fish protein hydrolysate on animal performance may be the high content of short peptides and free amino acids which are palatable and more readily absorbed than intact protein without preceding digestion by pancreatic proteases (Gilbert et al 2008).

The EE content in HWWE or HWWO was higher than that in fish meal or soya bean meal, which is a typical characteristic of catfish by-products in the Mekong Delta (Thuy et al 2007). EE digestibility showed higher values in HWWED or HWWOD diets, this could be explained by the feed characteristics, as the digestibility of dietary nutrients in poultry can be influenced by the characteristics of the feed, and the amount of digestible EE was relatively and positively related to the dietary EE content. In fact, the digestibility of EE in HWWOD or HWWED was higher than in the other diets, which can be explained by a high fat content in these diets as compared with the other diets. In addition, the catfish by-products has high fat which is rich of unsaturated fatty acids (Thuy et al 2007), and this may be responsible for high energy digestibility values and high digestibility of unsaturated fatty acid (Lesson and Summers 2001), because unsaturated fatty acids are more efficiently digested and absorbed than saturated fatty acids (Baiao 2005).

Apparent ileal digestibility and total tract retention of amino acids

The apparent ileal digestibility and total tract retention of amino acids are showed in Table 3. There was little higher in the digestibility values of amino acids in the excreta than that in the ileal digesta, it is agreement with Onimisi et al (2008) and Kadim et al (2002) who showed that there was significant differences between feacal and ileal digestibility of amino acid for animal meals, it may be due to the interferences in the gut of bird, the fermentation occurs in the caeca of poultry, which is likely to influence the amino acid contents of excreta and thus modify results for digestibility (Short et al 1999). Moreover, the less well balanced of amino acids in soya bean meal was the cause of lower AID and ATD of amino acids in the diets than fish meal and HWWE or HWWO.

In fact, Nguyen Thi Thuy and Nguyen Cong Ha (2016) showed that protein hydrolysate consists mainly of low molecular weight peptides which can be absorbed rapidly and have higher nutrient digestibility. Similar research from Rose Meire Vidotti et al (2003) also use sucessfully salmon by-product and hydrolysate by acidified to hydrolyse proteins into peptides and free amino acids, this can be used as feed for animal well and it is comparable with soya bean protein concentrate, fish meal. Hence, protein resources from Tra catfish processing waste water can be processed into protein hydrolysate for animal feed to improve absorb capacity, elucidate development capacity following the hydrolysis by enzyme and organic acid. And the results also showed that the hydrolysate of catfish by-product with enzyme or organic acid give similar quality then lead to similar in digestibility of nutrients and amino acids.


Supplementation of catfish by-product protein hydrolysate by enzyme or organic acid in the diets of Noi local chickens showed:


This research is funded by the Regional Program for Livestock-based Sustainable Agriculture in the Lower Mekong Basin (MEKARN II). Sincere thanks to Mr Manh housholder in Binh Thuy district for allowing us to carry out the experiment.


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Received 7 December 2016; Accepted 28 December 2016; Published 1 March 2017

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