Animal Feed Science and Technology 272 (2021) 114748

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Evaluation of pepsin derived tilapia fish waste protein hydrolysate

as a feed ingredient for silver pompano (Trachinotus blochii)
fingerlings: Influence on growth, metabolism, immune and
disease resistance
C.S. Tejpal a, *, P. Vijayagopal b, K. Elavarasan c, D.L. Prabu b, R.G.K. Lekshmi a,
R. Anandan a, E. Sanal b, K.K. Asha a, N.S. Chatterjee a, S. Mathew a, C.
N. Ravishankar a
a
Biochemistry and Nutrition Division, ICAR-Central Institute of Fisheries Technology, Cochin, 682029, India
b
Marine Biotechnology Division, ICAR-Central Marine Fisheries Research Institute, Cochin, 682 018, India
c
Fish Processing Division, ICAR-Central Institute of Fisheries Technology, Cochin, 682029, India

A R T I C L E I N F O A B S T R A C T

Keywords: Fish protein hydrolysate (FPH) has gained importance in recent times due to its growth and
Tilapia protein hydrolysate health promoting properties. Forty days feeding trial was carried out to study the effect of FPH
Fish growth prepared from tilapia fish processing waste (TFW) on growth, metabolism, immunity and resis­
Metabolism and immune response
tance against bacterial infection induced by Vibrio anguillarum in silver pompano (Trachinotus
Silver pompano (Trachinotus blochii) and Vibrio
anguillarum
blochii) fingerlings. During the study, fishes were fed practical diets containing 0, 30, 60 and 90 g/
kg tilapia protein hydrolysate (TPH) at 70 g/kg body weight per day initially which was gradually
reduced to 30 g/kg. Animals fed with 90 g/kg tilapia protein hydrolysate in diet showed signif­
icantly (P < 0.05) higher weight gain percentage, specific growth rate, feed conversion ratio and
protein efficiency ratio. Similarly, metabolic enzyme activities were significantly higher
(P < 0.05) in control group which decreased gradually with increasing level of TPH supple­
mented in diet. Significantly (P < 0.05) higher lysozyme activity and disease resistance against
Vibrio anguillarum was observed in the fishes fed with 90 g/kg tilapia protein hydrolysate in diet.
It can be concluded from the study that dietary supplementation of TPH shows promising results
in terms of improved growth, metabolism, immune response and resistance against bacterial
infection in silver pompano fingerlings.

Abbreviations: ALT, alanine amino transferase; ANFs, anti-nutritional factors; AST, aspartate amino transferase; CM, cumulative mortality; CRD,
completely randomized design; EAA, essential amino acids; FCR, feed conversion ratio; FPH, fish protein hydrolysates; GIFT, genetically improved
farmed tilapia; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; PB, phosphate buffer; PBS, phosphate buffer saline; PER, protein effi­
ciency ratio; RPS, relative percent survival; SGR, specific growth rate; SOD, superoxide dismutase; TPH, tilapia protein hydrolysate; TFW, tilapia fish
processing waste.
* Corresponding author.
E-mail address: tejpal.arun@rediffmail.com (C.S. Tejpal).

https://doi.org/10.1016/j.anifeedsci.2020.114748
Received 29 May 2019; Received in revised form 27 October 2020; Accepted 28 October 2020
Available online 2 November 2020
0377-8401/© 2020 Elsevier B.V. All rights reserved.
C.S. Tejpal et al. Animal Feed Science and Technology 272 (2021) 114748

1. Introduction

Global demand for fish is ever increasing due to beneficial aspects of fish on health and the demand is projected to be 215 mmt by
2050 (Bene et al., 2015). Similarly, in India the demand is projected to be around 20.00 mmt. It is a hard task to achieve the projected
growth without increasing the culture area and the efficiency of production. However, it can be achieved by making use of novel
strategies such as utilization of available low saline waters for fish farming with suitable candidate species and/or increasing the
stocking density.
In India, silver pompano (Trachinotus blochii) is considered to be a potential candidate for both marine as well as low saline water
farming since it can be acclimatized even at low salinity of about 8–10 g/kg. Fish is also well known for its faster growth rate and for
good nutritional quality of its meat. Locally, silver pompano is comparable with silver pomfret, in terms of body shape, colour pattern,
meat texture and quality. The culture practice of pompano has been successfully established in many Asia-Pacific countries particularly
in Taiwan and Indonesia. Pompano culture in India is at a nascent stage. The breeding and culture technique of pompano has been
established by the Central Marine Fisheries Research Institute, Kochi, India (CMFRI, 2014). On the other hand, intensive fish culture
requires high quality protein diet. Since many years fish meal is being used as a protein source in diet for fish due to several attributes
such as well-balanced essential amino acids (EAA), fatty acid molecules, high palatability and unidentified growth promoting sub­
stances (Hardy, 2010). The fast growth of aquaculture industry has resulted in high demand for fish meal over a period of time. At
present, the availability of fish for preparing fishmeal has substantially reduced with the cost of fishmeal going up.
Although, many plant protein sources have been investigated to substitute fishmeal in aqua-feed, nutritionists face difficulties in
aqua-feed formulation, mainly due to the presence of anti-nutritional factors (ANFs) and disproportionate amino acid composition
which affects the growth and immune response of fish. Emergent understanding on bioactive potential of enzymatically derived
peptides has led fish nutritionists to concentrate efforts towards inclusion of peptides with bio-functional capacities in feed formu­
lations (Kumar et al., 2014). The existing situation demands the utilization of available aquatic food processing waste through its
conversion into fish protein hydrolysate and its use as an alternative source of protein ingredients for aquaculture sector. In the recent
past, fish protein hydrolysate (FPH) has gained attention as a functional food ingredient and also has ample scope as a feed ingredient
(Khosravi et al., 2015).
Fish processing industry generates 60 % processing waste including head, skin, trimmings, fins, frames and viscera which can be
used as raw material for the production of FPH, which is a mixture of amino acids and peptides prepared through enzymatic hydrolysis
and akin to fish meal (Dekkers et al., 2011; Chalamaiah et al., 2012; Ibrahim et al., 2015; Tejpal et al., 2017). Fish protein hydrolysates
are found to have higher digestibility, easy assimilation and health promoting properties compared to fishmeal and the published
reports also highlight about the use of FPH as a fish feed ingredient in feed industry in terms of better growth, improved immunity and
efficient feed conversion (Tang et al., 2008; Zheng et al., 2012a, b; Niu et al., 2014). Khosravi et al. (2015) reported the influence of
krill, shrimp and fish protein hydrolysates as feed ingredients in the diet of juvenile red sea-bream on growth and disease resistance
against bacterial (Edwardsiella tarda) infection.
In recent times, much emphasis is given on farming of genetically improved farmed tilapia (GIFT) globally. The main processed
form of tilapia is filleting with yields of 300− 400 g / kg and the rest of the unutilized raw material accounts for about 600− 700 g / kg
(Clement and Lovell, 1994; Tejpal et al., 2017). The main objective of the study was to evaluate the effect of FPH a high value product
derived using pepsin, valorised from GIFT waste, on growth, metabolism, immunity and resistance to Vibrio anguillarum in the fin­
gerlings of a candidate mariculture species, Trachinotus blochii.

2. Materials and methods

2.1. Preparation of protein hydrolysates

Tilapia, Orechromis niloticus slaughter waste which comprises of head, skin, trimmings, fins, frames and viscera were collected from
a nearby fish market located at Thoppumpady, Cochin and fish processing pilot plant, ICAR-CIFT, Cochin, India. The quality of samples
were maintained using flake ice. The collected whole tilapia fish waste was rinsed in potable water and sliced. Further, it was mixed
with distilled water at a ratio of 1:2 and ground into paste using a household warring mixer (MX-AC350, Super Mixer Grinder,
Panasonic, Panasonic Appliances India Co., Ltd, India). The homogenate obtained was adjusted to pH 2.5 using 2 M HCl and pre
incubated at 37 ◦ C for 5 min to attain temperature equilibrium. Pepsin (from porcine gastric mucosa, powder, C 250 units/mg solid,
from Sigma-Aldrich, MO, USA) was added at an enzyme to substrate ratio of 10 g/kg (w/w) to initiate enzymatic hydrolysis. The

Table 1
Proximate composition of ingredients (g/kg) used for feed formulation.
Feed ingredients Tilapia fish waste Tilapia protein hydrolysate Soybean meal Shrimp meal Fish meal Wheat flour

Moisture 650.30 ± 7.51 50.49 ± 0.87 77.2 ± 11.92 60.0 ± 63.37 65.3 ± 7.77 8.73 ± 1.61
Crude protein 150.39 ± 2.60 820.0 ± 14.20 497 ± 25.45 616.1 ± 36.04 679.4 ± 4.37 138.30 ± 4.22
Crude lipid 60.09 ± 1.04 10.3 ± 0.18 7.68 ± 1.39 62.6 ± 1.32 93.46 ± 3.52 27.83 ± 4.81
Ash 80.6 ± 1.39 90.79 ± 1.57 79.6 ± 0.4 152.37 ± 5.33 119.4 ± 0.4 17.4 ± 3.62
Metabolizable energy (MJ/kg) – 12.94 11.85 12.67 13.92 11.66

Data given as expressed as mean of three replicates.

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C.S. Tejpal et al. Animal Feed Science and Technology 272 (2021) 114748

process was carried out for 3 h at 37 ◦ C and the reaction was terminated by heating the mixture in a boiling water bath for 15 min.
After cooling the mixture to room temperature, the pH was adjusted to 7 using 2 M NaOH. The mixture was filtered through a muslin
cloth and centrifuged (Thermo Fisher, Heraeus Multifuge 3SR+ Germany) at 10,000 rpm for 15 min to remove the fine solids. The
supernatant obtained was subjected to spray drying using a spray dryer (SM Scientech, SMST, Machine No.-16, India). Inlet tem­
perature, outlet temperature, and feeding rate were 180, 80 ◦ C and 20 rpm, respectively. (Tejpal et al., 2017). The hydrolysates ob­
tained were stored under desiccated condition till further use in feed formulation. The proximate composition of tilapia protein
hydrolysate (TPH) is presented in Table 1.

2.2. Experimental animals, design and diet preparation

Two-month old silver pompano Trachinotus blochii fingerlings were procured from marine fish hatchery, ICAR- Central Marine
Fisheries Research Institute, Mandapam, Tamil Nadu, India with average weight of fish ranging from 2.0 to 3.0 g. Fishes were
transported with sufficient oxygen enriched atmosphere in polythene bag. Upon arrival at the wet-lab, ICAR-CMFRI, Kochi, Kerala,
India, utmost care was taken while transferring the fish fingerlings to a circular tank (1-ton water holding capacity, salinity 20 g/kg)
and fishes were under observation for 12− 14 h. The stock was acclimatized for a period of 15 days under continuous aeration.
One hundred and twenty silver pompano fish fingerlings were distributed in to four experimental groups having 12 glass tanks with
75 L capacity. Ten numbers of fish were stocked in each replicate. Fishes were fed with TPH supplemented diet at different concen­
trations viz. 0, 30, 60 and 90 g/kg (TPH/Diet w/w). Completely Randomized Design was adopted to carry out the feeding trial for a
period of 40 days and the experimental groups were arranged in triplicate viz. TPH0 (0 g TPH/kg of feed); TPH30 (30 g TPH/kg of
feed); TPH60 (60 g TPH/kg of feed); and TPH90 (90 g TPH/kg of feed). The practical diet composition is given in Table 3. The source of
protein used was fish meal, shrimp meal and soybean meal. The lipid and carbohydrate sources used were fish oil and wheat flour,
respectively. The details of levels of TPH inclusion in feed are given in Table 3. Fish feed ingredients were thoroughly mixed with
water, followed by steam cooking for 10 min. After cooling, vitamin-mineral pre-mix was added to the dough. Feed was prepared by
using hand pelletizer and dried at 60 ◦ C in a hot air oven. During the experiment trial a constant natural photoperiod of 12 h light and
12 h dark was maintained. Continuous flow of water system (1.5 L–1) was used to maintain constant water level in the experimental
tanks and aeration was provided throughout the day. Initially, fishes were fed at 70 g/kg and gradually reduced to 30 g/kg of the body
weight for experimental period of 40 days (twice daily at 10:00 and 15:00 h). The unconsumed feed and faecal matter were siphoned
out regularly. All the water quality parameters were monitored daily during experimental trial: water temperature was maintained at
27− 29 ◦ C, dissolved oxygen at 5.5–6.5 mg L− 1, salinity was maintained at 17.5–20.5 g kg-1, pH at 7.5–7.8, and ammonia (NH+ 4 -N) at
<0.05 mg L− 1.

2.3. Proximate composition of tilapia waste, protein hydrolysates and major ingredients

Proximate composition of whole tilapia waste, prepared hydrolysates as well as major ingredients (fish meal, shrimp meal, soybean
meal and wheat flour) used in the feed formulation were determined as described in AOAC (2005). The dry matter (AOAC, 2005:
934.01) content of all the samples were estimated by hot air oven method by drying the samples to constant weight. Protein estimation
was carried out by determining the nitrogen content (AOAC, 2005: 988.05) and multiplying with a factor value of 6.25. Lipid

Table 2
Amino acid profiles of tilapia waste protein hydrolysates.
Essential amino acids (g/kg)

Arginine 42.12 ± 0.07

Histidine 19.14 ± 0.07
Methionine 10.88 ± 0.07
Isoleucine 14.01 ± 0.05
Lysine 26.26 ± 0.16
Leucine 36.45 ± 0.35
Phenylalanine 15.79 ± 0.06
Threonine 21.74 ± 0.15
Valine 26.29 ± 0.11
Tyrosine 10.49 ± 0.19
Total essential amino acids 212.67±0.54

Non-essential amino acids (g/kg)

Aspartic acid 60.63 ± 0.21

Glutamic acid 97.88 ± 0.12
Glycine 122.69 ± 0.49
Alanine 79.12 ± 0.28
Proline 41.81 ± 0.13
Cystine 1.10 ± 0.09
Serine 25.94 ± 0.41
Total amino acids 652.33±0.63

Note: Values are expressed as mean of three replicates.

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estimation (AOAC, 2005: 920.39) was carried out gravimetrically using petroleum ether as a solvent in Soxhlet apparatus. The ash
content of the samples was determined after incineration in a muffle furnace at 550 ◦ C for 6 h. Amino acid composition of tilapia
protein hydrolysate (TPH) was assessed using the method described by Ishida et al. (1981) and with slight modification as per Asha
et al. (2014) using LC-10AT vp high performance liquid chromatography (Shimadzu) with an ion exchange column and a fluorescence
detector. Prior to analysis, the column was equilibrated with the following running conditions which include: injection volume of
20 μl; mobile phase (A and B) A consisting of sodium citrate and ethanol (pH 3.5) and B, sodium citrate and NaOH (pH 9.8), with flow
rate of 0.4 mL/min, and the column temperature set at 60 ◦ C. Detection of fluorescence excitation and emission were at 340 and
450 nm, respectively. The samples were hydrolysed in 6 N HCl in evacuated sealed tubes at 110 ◦ C for 24 h. After derivatisation by
O-phthalaldehyde, amino acids were identified and quantified by comparison of their retention times with those of standards (Sigma)
and the data is given in the Table 2. The Metabolizable Energy content of the feeds were determined as described by NRC (2011),
ME = DE - (ZE + UE), where ME: metabolizable energy; ZE: branchial energy loss and UE: urine, urinary energy loss.

2.4. Growth performance

The growth performance of fingerlings was assessed in terms of weight gain, specific growth rate (SGR), feed conversion ratio (FCR)
and protein efficiency ratio (PER), as given below. Initial weight of the fishes was recorded and during the experimental period, fishes
were weighed at 7-day interval.

Weight gain (%) = 100 x (FW – IW)/IW

Specific growth rate (SGR) = 100 x (ln of FW – ln of IW)/ number of culture days

Feed conversion ratio (FCR) = F / (FW – IW)

Protein efficiency ratio (PER) = Wet weight gain / crude protein fed

Total feed intake = {(% body weight of feed offered * weight of fish (g) / 100} x Number of days of feeding trial.

Where IW and FW are initial and final weight (g) of fish and F is the total feed given to fish (g).

Table 3
Experimental diets for silver pompano (Trachinotus blochii).
Experimental diets
Ingredients (g/kg, as fed)
TPH0 TPH30 TPH60 TPH90
1
Soybean meal 95 95 75 75
Shrimp meal2 310 290 280 260
Wheat gluten3 30 30 30 30
Gelatin4 20 20 20 20
Fish meal5 310 290 280 260
Tilapia protein hydrolysate 00 30 60 90
Wheat flour6 143 153 163 173
Vitamin C7 10 10 10 10
BHT 8 2 2 2 2
Fish oil9 50 50 50 50
Soya lecithin10 10 10 10 10
Vitamin + mineral mixture11 20 20 20 20

Chemical composition (g/kg, as fed)

Metabolizable energy12 (MJ/kg) 14.38 14.39 14.63 14.45
Dry matter 898 897 900 896
Crude protein 400 402 405 404
Crude lipid 83 89 91 84
Ash 97 103 94 92

*TPH, Tilapia protein hydrolysate.

TPH0, 0 g TPH/kg of feed); TPH30, (30 g TPH/kg of feed); TPH60, (60 g TPH/kg of feed); and TPH90, (90 g TPH/kg of feed).
Note: The ingredients 1–11 were purchased from the following suppliers: 1SakthiSoya, Coimbatore, India,2KhajaMohideen, Wall Tax Road, Chennai,
India, 3Viveka Essence Mart, Wall Tax Road, Chennai, 4Gelatin: (96 % CP) Himedia Ltd., India, 5Raj Fishmeal and Oil Co., Malpe, Mangalore, India, 6
From local market, Kochi, India, 7STAY-C DSM Nutritional technologies, Mumbai,India, 8Butylated hydroxyl toluene (Assay 99 %): SD Fines Chemical
Ltd. India, 9Crude sardine oil from Kiriyathan Trading Co., Kochi, India, 10Ocean Nutrition International LLC, USA and 11Vitamin + mineral mixture
(Supplevite®): composition per kg diet: vitamin A-24 mg, vitamin D3–0.2 mg, vitamin B2–16 mg, vitamin E-5.4 mg, vitamin K-8 mg, Calcium
pantothenate- 20 mg, Nicotinamide- 8 mg, vitamin B12–48 mg, choline chloride − 1200 mg, calcium − 6000 mg, manganese-2200 mg, iodine-8 mg,
iron-60 mg, Zinc -120 mg, copper-16 mg, cobalt -3.6 mg., Zydus Animal Health, Gujarat, India.

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2.5. Sampling procedure and sample preparation for enzyme assay

After the feeding trial, pooled blood of two fishes per individual tank was used to collect serum (totally 6 fishes were used for
collection of blood in each treatment). Further, randomly three fishes per tank were used to collect muscle, liver and gill tissue samples
(totally 9 fishes per treatment were used to collect the tissue sample). Fishes were captured smoothly and anaesthetized by clove oil
(50 μl/L). Blood was collected from the caudal vein, and transferred to Eppendorf tubes which were kept on ice till the serum was
collected. With the help of a mechanical tissue homogenizer, the tissues were homogenised with pre-chilled sucrose solution (0.25 M).
Homogenized tissue samples were centrifuged (5000 x g at 4 ◦ C for 10 min); supernatants were collected and preserved at -20 ◦ C.

2.6. Enzyme assays

Pooled tissue samples such as muscle, liver and gill from individual tanks were subjected to enzyme assay viz. aspartate amino
transferase (AST) and alanine amino transferase (ALT) activities were measured as described by Wooten (1964). Lactate dehydro­
genase (LDH) and malate dehydrogenase (MDH) activities were measured as described by Wroblewski and Ladue (1955) and Ochoa
(1955) respectively. Superoxide dismutase (SOD) enzyme activity was estimated by the method of Misra and Fridovich (1972).

2.7. Lysozyme activity

Pooled serum collected from each tank was assayed for serum lysozyme activity of Trachinotus blochii fingerlings using lysozyme
assay kit (Bangalore Genei, India) as described by Jha et al. (2007). Serum samples were initially diluted with PB (phosphate buffer; pH
7.4) in such a way that a final concentration of 0.33 mg/mL is attained. Bacterial suspension (Micrococcus luteus) was prepared with
phosphate buffer (A450 = 0.5− 0.7). Exactly, 3 mL of bacterial suspension was pipetted out in sample cuvette and 50 μL serum sample
was added. The reaction mixture was thoroughly mixed for 15 s and absorbance was noted at 450 nm using a spectrophotometer. The
absorbance was compared with standard lysozyme of known activity and expressed as U/min/mg protein.

2.8. Challenge study with Vibrio anguillarum

On completion of feeding trial, fishes (5 fish per tank) were subjected to challenge study with virulent Vibrio anguillarum obtained
from Fish Health and Microbiology laboratory, Marine Biotechnology Division, CMFRI. Under controlled environment, the bacteria
were cultured in tryptone soya broth (Himedia, India) at 30 ◦ C for 24 h and the broth cultures were harvested by centrifuging the
culture broth at 10,000 rpm for 10 min at 4 ◦ C. The supernatant was discarded and the pellets were washed in sterile PBS (Phosphate
Buffer Saline; pH 7.2) for 3 times. The bacterial concentration was calculated by measuring absorbance using a spectrophotometer and
also was confirmed using plate count method. The final bacterial concentration was maintained at 107 CFU/mL by serial dilution. After
40 days of feeding trial, precisely, 0.2/0.1 mL of bacterial suspension was injected intraperitoneally to the fishes (Ranjan et al., 2014).
Survival of fishes was observed for 10 days. Cumulative mortality and relative percentage survival (RPS) were calculated as follows:

Cumulative Mortality (%) = (total mortality in each treatment after challenge / total number of fish challenged for same treatment) X 100

RPS = {(mortality (%) of control – mortality (%) of treatment) / mortality (%) of control} X 100

2.9. Statistical analysis

All data were subjected to one-way ANOVA and a contrast analysis was performed by taking both linear and quadratic orthogonal
polynomials. The proximate analysis of feed ingredients and diets were carried out with three individual replicate. Data on growth
performance, enzyme activities and cumulative mortality/survival rate were analyzed using the tank as the experimental unit. In cases
where both the linear as well as quadratic effects were significant, a second order model of the following form was fitted

f (xu ) = β0 + β1 xu + β2 xu2 + eu

where u = 1, 2,…,N, xu is the level of FPH in the uth treatment, f(xu) denotes the response obtained from uth treatment and eu is the
random error associated with the uth observation that is independently and normally distributed with mean zero and common
variance σ2, β0 is the intercept, β1 and β2 are the linear and quadratic regression coefficients respectively. Adequacy of the fitted model
was assessed by R2. The level of significance was set at P < 0.05. The statistical analyses were performed using SAS 9.4 (SAS, 2016)
software for Windows.

3. Results

3.1. Proximate composition of tilapia waste, protein hydrolysates and major ingredients and feed

Proximate composition of the ingredients used such as tilapia protein hydrolysate, soybean meal, shrimp meal, fish meal, wheat

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flour, tilapia fish processing waste and the diet prepared are given in Table 1 and 3. Crude protein content of the diets prepared ranged
from 446.0 to 450.7 g/kg of feed. Among the feed ingredients, highest protein content was recorded in the tilapia fish protein hy­
drolysate, followed by fish meal, shrimp meal, soybean meal, tilapia fish waste and wheat flour.
The total amino acid content of spray dried FPH prepared from tilapia fish waste was 652.33 g/kg of hydrolysate and the total
essential amino acid content was 212.67 g/kg. Glycine content was found to be the highest (122.69 g) followed by glutamic acid
(97.88 g). Arginine content was found to be the highest among essential amino acids (42.12 g). The limiting amino acid in FPH sample
was cysteine. The results of amino acid analyses is presented in Table 2.

3.2. Growth performance

The initial weight, final weight, feed intake, weight gain, FCR, SGR and PER are shown in Table 4. Linear contrasts showed sig­
nificant (P < 0.05) effect of inclusion of TPH at graded level among the control and treatment groups. Animals fed with TPH90
recorded the highest weight gain (141.93), SGR (1.47), PER (1.96) and FCR (1.49) than the other treatment groups.

3.3. Enzyme assays

3.3.1. Aspartate amino transferase (AST) and Alanine amino transferase (ALT)
Results of AST and ALT activities of different tissues viz. muscle, liver and gill are given in Table 5. AST and ALT activities were
found to be significantly (P < 0.05) different in the linear effect among the treatments, control group recorded higher enzyme activity
than the treatment groups. Proportionate inclusion of TPH in the diet of the treatment group had shown decreased AST and ALT
activities. Gill and muscle tissues had the highest AST and ALT activities, respectively. AST activity in gill and ALT activity in liver
showed significant (P < 0.05) differences with respect to the quadratic effect. The best fit equations demonstrating the relationships of
enzyme parameters in silver pompano (f(xu)) with tilapia fish waste protein hydrolysate (xu) can be represented in equations as
follows (i), (ii). The optimum values of the parameters and their responses are given in Table 6.

AST Gill, f (xu ) = 65.64 − 1.02xu + 0.006x2u (i)

ALT Liver, f (xu ) = 19.90 − 0.26xu + 0.00001x2u (ii)

3.3.2. Lactate dehydrogenase (LDH) and Malate dehydrogenase (MDH)

Data pertaining to the LDH and MDH activities were recorded and presented in the Table 5. Similar to AST and ALT activities,
tilapia protein hydrolysate (TPH) supplementation in diet has recorded significant (P < 0.05) difference with linear effect and reduced
level of LDH and MDH activities in liver and gills. However, LDH and MDH activities of the muscle tissue did not show any difference
between the treatment groups fed with 30 and 60 g TPH/kg in the diet. Higher LDH and MDH activities was recorded in the muscle
followed by gills and liver. LDH activity in the liver was significantly (P < 0.05) different with respect to the quadratic effect. The best
fit equation with respect to the quadratic trend of LDH activity of liver can be represented as:

LDH Liver, f (xu ) = 3.77 − 0.07xu + 0.00041x2u

3.3.3. Superoxide dismutases (SOD)

Activity of antioxidant enzyme, SOD in different tissues (muscle, liver and gills) of control and treatment groups is presented in
Table 5. With increase in TPH supplementation from 0 (TPH0) to 90 g TPH/kg of feed, SOD activity in liver and muscle showed
significant (P < 0.05) difference with linear effect, whereas, SOD activity in gills was found to be significant with linear and quadratic
effect (Table 5). Liver exhibited highest SOD activity followed by gills and muscle. The best fit equation depicting the quadratic trend of

Table 4
Growth performance, feed intake, feed efficiency and protein efficiency ratio of silver pompano fingerlings fed experimental diets for 40 days.
Treatment TPH0 TPH30 TPH60 TPH90 SEM P-value (linear effect) P-value (quadratic effect)

Initial weight (g) 3.07 2.93 3.03 2.95 0.025 0.230 0.567
Final weight (g) 5.90 6.40 6.80 7.13 0.173 0.005 0.730
Weight gain (g) 2.83 3.47 3.77 4.18 0.181 0.004 0.668
Total Feed intake (g feed/fish) 6.44 6.16 6.37 6.20 0.052 0.959 0.400
Weight gain% 92.36 118.41 124.23 141.93 6.479 0.003 0.630
FCR 2.37 1.78 1.70 1.49 0.118 0.010 0.468
PER 1.27 1.63 1.72 1.96 0.088 0.005 0.850
SGR 1.08 1.30 1.34 1.47 0.060 0.004 0.530

TPH, tilapia protein hydrolysate.

1. TPH0 = (0 g TPH/kg of feed); TPH30 = (30 g TPH/kg of feed); TPH60 = (60 g TPH/kg of feed); and TPH90 = (90 g TPH/kg of feed).
Weight gain%= (final weight-initial weight) X 100/ initial weight; Total Feed intake (g feed/fish) = {(% body weight feed offered * weight of fish (g) /
100} X Number of feeding trial; Feed conversion ratio (FCR)= Total feed given on dry weight (g) / Weight gain on wet weight (g); Protein efficiency
ratio (PER) = Total wet weight gain (g) / Crude protein fed (g); Specific growth rate (SGR) = (Loge average final weight - loge average initial weight)
× 100 / number of days and SEM = standard error of the mean.

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Table 5
Enzyme activities of silver pompano fingerlings fed the experimental diets for 40 days.
Parameters Tissue Treatments
TPH0 TPH30 TPH60 TPH90 SEM P-value (linear effect) P-value (quadratic effect)

Muscle 15.53 9.77 7.586 5.03 0.920 <0.001 0.122

AST Liver 20.71 14.83 8.23 4.40 1.450 <0.001 0.500
Gill 67.80 34.24 33.83 23.52 3.800 <0.001 0.005
Muscle 38.40 30.50 25.166 23.66 1.630 <0.001 0.101
ALT Liver 20.25 12.21 9.75 5.97 1.110 <0.001 0.004
Gill 22.27 16.35 6.04 3.18 1.730 <0.001 0.351
Muscle 9.82 7.61 7.68 6.95 0.330 <0.001 0.061
LDH Liver 3.86 1.78 1.14 0.82 0.279 <0.001 <0.001
Gill 6.60 5.98 3.89 2.11 0.478 <0.001 0.392
Muscle 134.25 128.41 125.56 101.05 3.500 0.002 0.073
MDH Liver 62.68 51.52 40.90 28.26 3.030 <0.001 0.815
Gill 129.84 114.60 92.70 63.47 6.266 <0.001 0.361
Muscle 44.14 37.84 38.09 36.10 1.120 0.001 0.050
SOD Liver 155.53 141.96 141.98 132.68 9.688 0.009 0.640
Gill 59.13 45.46 43.20 36.87 3.310 0.057 0.007
Lysozyme activity Serum 209.84 288.52 314.75 413.11 22.250 <0.001 0.310

1. Data presented as means of five replicates.

2. TPH0, (0 g TPH/kg of feed); TPH30, (30 g TPH/kg of feed); TPH60, (60 g TPH/kg of feed); and TPH90, (90 g TPH/kg of feed).
3. AST, Aspartate amino transferase, Unit: nano moles oxalo acetate released/ mg protein/ minute at 37 ◦ C; ALT, Alanine amino transferase, Unit:
nano moles of sodium pyruvate formed/mg protein/minute at 37 ◦ C; MDH, Malate dehydrogenase, Unit: nanomoles of Oxaloacetate utilized /mg
protein/minute; LDH, Lactate dehydrogenase, Unit; nanomoles of pyruvate utilized//mg protein/minute; SOD, Superoxide dismutase, Unit = 50 %
inhibition of epinephrine auto oxidation per mg protein / min and Lysozyme activity expressed in (U/min/mg protein).

Table 6
Parameter estimates of second order model fitted for enzyme parameters in silver pompano obtained by canonical analysis.
Responses

AST Gill ALT liver LDH liver SOD Gill

β0 65.64* (3.35) 19.90* (0.71) 3.77* (0.17) 57.49* (3.98)

β1 − 1.02* (0.18) − 0.26* (0.04) − 0.07* (0.009) − 0.73* (0.21)
β2 0.006* (0.002) 00,001* (0.0004) 0.00041* (0.00009) 0.007* (0.002)
R2 0.80 0.91 0.89 0.58
Optimum value 79.40 108.91 83.01 53.48
Response at optimum activity 24.94 5.89 0.90 38.01

Values in parentheses indicate SE of estimates.

*
indicate significance at P < 0.05.

SOD activity in gills can be represented as follows:

SOD Gill, f (xu ) = 57.49 − 0.73xu + 0.007xu2

Table 7
The cumulative mortality (%) and relative percentage of survival (%) of silver pompano fingerlings fed graded level of TPH for 40 days and post
challenged by Vibrio anguillarum 10 days.
Parameters TPH0 TPH30 TPH60 TPH90 SEM P-value (linear effect) P-value (quadratic effect)

Cumulative mortality (%) 73.33 40.00 26.66 13.33 7.961 0.002 0.350
Relative Percentage of Survival (%) – 43.77 60.69 77.62 8.588 0.002 0.305

TPH, tilapia protein hydrolysate.

1. Data presented as means of five replicates.
2. In cases where only the linear effect is significant (P < 0.05), alphabetical letter grouping is followed. Means with different superscripts in the same
row indicate significant differenceTPH0, (0 g TPH/kg of feed); TPH30, (30 g TPH/kg of feed); TPH60, (60 g TPH/kg of feed); and TPH90, (90 g TPH/
kg of feed).
3. Cumulative mortality (%) was calculated as per equation [Cumulative mortality (%) = (total mortality in each treatment after challenge / total
number of fish challenged for same treatment) X 100] and Relative percentage of survival (%) was calculated as per the following equation [RPS =
{(mortality (%) of control – mortality (%) of treatment) / mortality (%) of control} X 100] by taking into account the percentage of survival in each
treatments.

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C.S. Tejpal et al. Animal Feed Science and Technology 272 (2021) 114748

3.4. Lysozyme activity

The lowest lysozyme activity of 209.84 was recorded for control group and the highest activity was recorded for the treatment
group fed with 90 g TPH/kg of feed. Lysozyme activity in fishes fed on TPH30 and TPH60 had no significant difference between them
(Table 5).

3.5. Disease resistance

During 40 days of feeding trial, 100 percent survival of fish was recorded in all the groups. After the main feeding trial, fishes (5 fish
per tank) were subjected to challenge study in order to know the efficiency of TPH as an immune booster. In the challenge study, all the
fishes including control and treatment groups were injected with Vibrio anguillarum at known concentration and animals were kept
under observation for 10 days. The clinical signs observed in the fish were hyperaemia on the eyes, lethargy and red spots on the
ventral side and swollen abdomen. These signs are typically associated with Vibrio anguillarum infection. The results presented in
Table 7 are linearly significant. Control group had 73.33 % of cumulative mortality and in treatment groups it was found to be
decreased to 40, 26.66 and 13.33 %. The relative percentage survival increased proportionately with inclusion level of TPH in the diet.

4. Discussion

Proximate composition of tilapia fish protein hydrolysate, fish meal, shrimp meal, soybean meal, tilapia fish waste and wheat flour
and the diets prepared are in agreement with those reported elsewhere (Khosravi et al., 2015; Haghbayan et al., 2015; Hamidoghli
et al., 2020). Well-balanced essential amino-acid profile of dietary protein source is the most crucial factor for optimum animal growth.
The amino acid profile of FPH is important in dictating the functional and health-promoting properties. Amino-acids are essential for
protein synthesis and act as carriers for the structural proteins, enzymes, carbon dioxide, oxygen and vitamins. The amino acid profile
of FPH varies with the specificity of enzyme used for digestion, nature of substrate and extent of hydrolysis. Higher glycine content of
hydrolysate obtained in this study can be attributed to the fact that the fish waste used for hydrolysis included skin which is rich in
collagen, a glycine rich protein. Tilapia waste hydrolysate was found to have good amount of essential as well as non-essential
amino-acids which are important to maintain the well-being of animal (Chalamaiah et al., 2012; Elavarasan and Shamasundar,
2016; Tejpal et al., 2017). Further, fish protein hydrolysates were found to have bio-functional properties like antioxidant, antimi­
crobial, and anti-inflammatory and can serve as food preservatives or as an alternative bioactive compound for synthetic antimicrobial
and antioxidants and anti-inflammatory compounds (Da Rocha et al., 2018). Srikanya et al. (2017) have demonstrated the preparation
of fish protein hydrolysate from Tilapia fish waste, and also indicted the possible use of FPH in different food system.
The data obtained from all the responses in this study were subjected to one-way ANOVA and contrast analysis by considering both
linear and quadratic orthogonal polynomials. Among the data analysed, parameters such as AST gill, ALT liver, LDH liver and SOD
activities of gill were found to follow a quadratic trend, and a second order model was fitted after canonical analysis for those pa­
rameters. Dietary supplementation of TPH significantly influenced the overall growth performance of silver pompano fingerlings.
Lowest FCR was recorded in the group fed 90 g TPH/kg of feed (TPH90) in the diet. Results indicate that food conversion was effective
with the inclusion of TPH proportionately. This is due to easy assimilation of the smaller size of peptides and free amino-acids.
Increased PER, SGR and weight gain could be explained in a similar manner. Tang et al. (2008) reported better growth perfor­
mance in the fishes supplemented with 100 g FPH/kg of feed. Silva et al. (2017) reported that inclusion of fish protein hydrolysate at
4.75 g/kg diet showed better growth performance in post-larval stage Nile tilapia. The presence of small peptides and their distribution
in FPH seem responsible for the improved performance in terms of growth in the fishes (Bilinski, 1974; Aksnes et al., 2006a, b;
Kotzamanis et al., 2007; Zheng et al., 2012a, b; Khosravi et al., 2015). Recent study revealed that dietary supplementation of protein
hydrolysate prepared from by-products of common carp at 50 g/kg feed level has showed improved growth in zebrafish (Zamor­
a-Sillero et al., 2019). Wei et al. (2017) reported that juvenile turbot (Scophthalmus maximus L.) fed with different molecular weight
FPH was found to have metabolite changes in liver and muscle which are associated with growth of fish.
In teleosts, energy is mainly derived from amino-acids (Demeal, 1978). Chatterjee et al. (2006) reported that the proteins and lipids
stored in fish will be utilized prior to carbohydrates. During stress, gluconeogenesis pathways get activated by cortisol (stress hor­
mone), stored protein and lipid are mobilized for the synthesis of glucose. The substrates for gluconeogenesis are the TCA cycle in­
termediates which are formed by the deamination of amino acids. In silver pompano fingerlings, the impact of incorporating tilapia
protein hydrolysate (TPH) in the diet, on tissue specific amino transferases, AST and ALT activities was studied. Higher AST and ALT
activities were observed in the muscle than that of liver and gills. The activities of AST in gills and ALT in liver of experimental fish
followed a quadratic trend, whereas their activity in other tissues analysed followed a linear pattern with respect to TPH levels. A
gradual decrease in AST and ALT activities in TPH fed animals reduced amino-acid mobilization for the production of glucose via
gluconeogenesis. Higher glucose production would result in reduced growth due to the greater mobilization of protein/amino-acids.
This is well correlated with the data obtained in the present study on growth related parameters. Earlier studies have correlated lower
AST and ALT activities with enhanced growth in fish and rat (Murray et al., 2000; Tejpal et al., 2009; Taha et al., 2014). In accordance
with our study, lower AST and ALT activities were recorded in the Juvenile Olive Flounder (Paralichthys olivaceus) fed with different
protein hydrolysates (Khosravi et al., 2018).
Conversion of lactate to pyruvate by LDH is catalysed with the help of coenzyme NADH. Thus LDH regulates glycolysis cycle and
supplies NAD+ (Grigo, 1975). Similarly, MDH converts the malate to oxaloacetate, using the electron acceptor, NAD+. Malate de­
hydrogenase helps to interconvert oxaloacetate to malate in mitochondria and malate to oxaloacetate in cytoplasm (Das et al., 2006).

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The activities of LDH and MDH are expected to increase during gluconeogenesis process. In our study, a quadratic trend was observed
for LDH activity of liver of fish with respect to the levels of dietary TPH, while the activity of LDH in muscle and gill tissues followed a
linear pattern with dietary TPH levels. The activity of MDH followed a linear trend in all the tissues analysed. The control group of the
present study showed higher MDH and LDH activities while the treatment groups showed lower activities. Increase in the activities of
metabolic enzymes (LDH and MDH) in the experimental animal can be ascribed to the production of desired substrate for gluco­
neogenesis (Das, 2002; Taha et al., 2014). Higher MDH activity in control shows that there is a huge need for energy and as a result,
more energy in produced through TCA cycle (Saurabh and Sahoo, 2008). Lower LDH and MDH activities in different tissues from the
treatment groups suggest that the supplementation of tilapia protein hydrolysate (TPH) may help to maintain normal metabolic
homeostasis in Trachinotus blochii fingerlings and support better growth. Gora et al. (2018) reported lower LDH and MDH activities in
Labeo rohita fingerings fed with graded level of fucoidan.
SOD converts superoxide radicals into molecular oxygen or hydrogen peroxide. The severity of damage to the cell or bio-molecules
by hydrogen peroxide is less compared to superoxide radicals. Free radicals generated are neutralized by SOD activity by which the
cells are protected from damage. When the animal is under stress due to unfavourable environmental conditions, levels of SOD
expressed are high. In the present study, antioxidant enzyme (SOD) activity was high in control group and low in the treatment groups
in a dose dependent manner. Peptides present in the fish protein hydrolysate (FPH) have been reported to have radical scavenging
properties and are also found to survive in gastrointestinal path (Elavarasan and Shamasundar, 2016). Hence, it is believed that the
tilapia protein hydrolysate (TPH) in diet scavenges free radicals generated during normal metabolism, which in turn minimizes the
necessity of expression of SOD in different tissues. Dietary supplementation of probiotics, prebiotics and immunostimulants at different
concentrations can lower or maintain stable SOD activity in treatment groups at par with the control group fed with basal diet (Mona
et al., 2015). Dietary supplementation of 1 and 3 % of fucoidan has showed lower SOD activity in liver and gill tissue (Gora et al.,
2018). In our study, the SOD activity of gills followed a quadratic trend, while that of muscle and liver a linear trend was observed. The
elevated levels of SOD activity may be correlated with oxidative stress response of the animal. Similarly, lower level of antioxidant
enzyme (SOD) activity was recorded in the treatment group fed with thiamine and pyridoxine incorporated vanillic acid-chitosan
(Tejpal et al., 2017).
Innate immune system in fish is a front line defence mechanism against a wide range of pathogens and the most important index of
innate immunity of fish is lysozymal activity (Medzhitov and Janeway, 1997; Ai et al., 2004). In the present study, fish fed with TPH
showed higher lysozyme activity than control group. Similarly, Tang et al. (2008) recorded higher lysozyme activity in Pseudosciaena
crocea R. fed with dietary FPH. Earlier studies have indicated a positive correlation between lysozyme activity and supplementation of
ascorbic acid (Roberts et al., 1995; Ortuno et al., 1999; Ai et al., 2006). In support to our study, Khosravi et al. (2018) reported that
dietary supplementation of tilapia hydrolysate or krill hydrolysate in low fishmeal diet has significantly enhanced the serum lysozyme
activity in juvenile Olive Flounder (Paralichthys olivaceus). The higher lysozyme activity in treatment group may also be related to the
higher disease resistance observed in the present study against Vibrio anguillarum.
Dietary supplementation of tilapia protein hydrolysate (TPH), improved disease resistance of silver pompano fingerlings against
Vibrio anguillarum. Peptides present in FPH showed antibacterial activity. FPH fed fish groups showed increase in serum lysozyme
activity. Lysozyme production is associated with a well-known disease fighting mechanism. Galaz et al. (2010) have reported disease
resistance in parrot fish against Vibrio anguillarum infection when diets were supplemented with Vitamin E. Similarly, supplementation
of immunostimulants like yeast extract, brewer’s yeast and spirulina enhanced resistance against bacterial (Aeromonas hydrophila)
infection in rohu fingerlings (Andrews et al., 2011). Siddik et al. (2018) reported that dietary supplementation of tuna hydrolysate at
61 g/kg diet showed disease resistance against Streptococcus iniae in juvenile barramundi (Lates calcarifer). Dietary supplementation of
protein hydrolysate in partial replacement of fishmeal exhibited improved disease resistance against Edwardsiella tarda in juvenile
Olive Flounder (Khosravi et al., 2018).

5. Conclusion

The eco-friendly approach of valorising fish processing waste to protein hydrolysates has far reaching impacts including organic
solid waste reduction and production of protein rich fish feed ingredient. The supplementation of protein hydrolysate prepared from
tilapia waste has showed enhanced growth performance in terms of the weight gain, specific growth rate, feed conversion ratio and
metabolic response in a dose dependent manner. Inclusion of tilapia protein hydrolysate in the diet has significantly enhanced the
innate immunity, antioxidant enzyme defence systems and also improved the disease resistance of Trachinotus blochii fingerlings
against Vibrio anguillarum. Hence, it is inferred that protein hydrolysate from tilapia fish waste could be a potential fish feed ingredient
in aqua-feed formulation. Further optimization on the inclusion levels of fish protein hydrolysate based on the species as well as life-
stage of fish aids to boost its utility in aqua feeds.

Ethical approval

“The experiment was subjected to internal Ethical review by a committee formed by the Marine Biotechnology Division, Central
Marine Fisheries Research Institute, Cochin, India. The study was conducted by following institutional guidelines for the care and use
of animals.”

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Declaration of Competing Interest

Authors have no conflict of interest.

Acknowledgements

The authors are thankful to the Director, ICAR- Central Institute of Fisheries Technology, Cochin and the Director, Central Marine
Fisheries Research Institute, Cochin for providing the necessary facilities and support. The authors thankfully acknowledge Dr. Eldho
Varghese, Scientist, CMFRI for his contribution in statistical analysis.

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