Ann. Anim. Sci., Vol. 21, No. 2 (2021) 575–587 DOI: 10.

2478/aoas-2020-0085

Replacement of fish meal with defatted and fermented

soybean meals in pompano Trachinotus blochii
(Lacepède, 1801) diets

Hung Phuc Nguyen1♦, Thinh Van Do2, Hau Duc Tran3, Trung Thanh Nguyen4

1
Department of Human and Animal Physiology, Faculty of Biology,
Hanoi National University of Education (HNUE), Caugiay 11310, Hanoi 10000, Vietnam
2
Centre for Aquaculture Biotechnology, Research Institute for Aquaculture No. 1 (RIA 1),
Tuson 16352, Bacninh 16000, Vietnam
3
Department of Zoology, Faculty of Biology, Hanoi National University of Education (HNUE),
Caugiay 11310, Hanoi 10000, Vietnam
4
Research Center for Aquafeed Nutrition and Fishery Post Harvest Technology,
Research Institute for Aquaculture No. 2 (RIA 2), District 1 71007, Ho Chi Minh City 70000, Vietnam
♦
Corresponding author: hungnp@hnue.edu.vn

Abstract
This study was conducted to examine the effects of the replacement of fish meal with defatted and
fermented soybean meals on the growth performance, apparent nutrient digestibility, bile acid
levels, and digestive enzyme activity of pompano Trachinotus blochii (Lacepède, 1801). Four diets
were formulated to replace 40% of fish meal with defatted soybean meal (SBM), SBM fermented
by Bacillus subtilis TH2 (FSBM1) or SBM fermented by B. subtilis B3 (FSBM2). The diets are
denoted as follows: FMD (fish meal-based diet, used as a Control), SBMD (fish meal replaced by
SBM diet), FSBM1D (fish meal replaced by FSBM1 diet), and FSBM2D (fish meal replaced by
FSBM2 diet). Thirty fingerling pompanos with an initial body weight of 15.3 ± 0.3 g were allocated
to each of eight indoor polyvinyl chloride tanks (500 L capacity), with two replicate tanks per
dietary treatment. For 8 weeks, fish were hand-fed the experimental diets to apparent satiation
twice daily. The final body weight, weight gain, specific growth rate, and feed conversion ratio of
fish fed SBMD and FSBM1D were significantly inferior to those in fish fed FMD (P<0.05). These
parameters were not significantly different between the FSBM2D and FMD experimental groups
(P>0.05). Fish fed SBMD showed significantly lower plasma total cholesterol, whole body lipids,
intestinal total bile acids and lipase activity than those fed FMD, whereas no significant differences
were observed among fish fed FSBM1D, FSBM2D, and FMD. Trypsin activity and protein ap-
parent digestibility coefficient were not significantly affected by the experimental diets. However,
lipid apparent digestibility coefficient was significantly lower in fish fed SBMD and FSBM1D
rather than FMD. There was no significant difference in lipid apparent digestibility coefficient be-
tween the FMD and FSBM2D experimental groups. The results indicated that SBM decreased bile
acid levels, lipase activity, lipid digestibility, and growth performance in pompano fish. These
parameters were increased by the inclusion of B. subtilis B3 fermented SBM in the diet, thus sug-
576 H.P. Nguyen et al.

gesting that fermentation of SBM with B. subtilis B3 may be an effective way to improve bile acid
levels, lipase activity, lipid digestibility, and growth performance of pompano fed an SBM-based
diet.

Key words: soybean meal, solid state fermentation, growth performance, digestibility, pompano
Trachinotus blochii

Fish meal is an important protein source in the aquafeed industry, but its mar-
ket price has been rising because of the expansion of aquaculture and the limited
availability of fish meal (Olsen et al., 2012; Hua et al., 2019). Defatted soybean
meal (SBM) has been considered to be the most cost-effective alternative to fish
meal because of its high protein content, relatively well-balanced amino acid profile,
and reasonable price (Storebakken et al., 2000; Porter and Jones, 2003). However,
when given diets containing more than 30% SBM, carnivorous species such as yel-
lowtail Seriola quinqueradiata (Shimeno et al., 1992), Atlantic salmon Salmo salar
(Krogdahl et al., 2003; Refstie et al., 2005), sharpsnout seabream Diplodus puntazzo
(Hernandez et al., 2007), rainbow trout Oncorhynchus mykiss (Romarheim et al.,
2008), and tiger puffer Takifugu rubripes (Lim et al., 2011) show a decreased growth
performance and feed utilization. In addition, high levels of SBM in fish feed induce
physiological digestive disorders such as hypocholesterolemia, inferior bile acid lev-
els, and poor dietary nutrient digestibility (Olli and Krogdahl, 1995; Takagi et al.,
2002; Nguyen et al., 2011 b, 2017; He et al., 2020). Anti-nutritional factors (ANFs)
in SBM, such as glycinin, β-conglycinin, trypsin inhibitors, raffinose and stachyose,
saponins, lectins, and phytate, are important factors responsible for these negative
effects in fish (Refstie et al., 1998; Francis et al., 2001; Iwashita et al., 2008; Li et
al., 2017 a, b).
Solid state fermentation has been suggested as an economical and effective meth-
od to reduce ANFs and increase the nutritional value of SBM. Fermentation has been
reported to decrease the amount of ANFs such as oligosaccharides, soy antigens, and
trypsin inhibitors in SBM (Refstie et al., 2005). Moreover, fermentation can also
break down glycinin and β-conglycinin, the major antigenic proteins in soybean, into
smaller peptides (Hong et al., 2004; Feng et al., 2007). Some studies have demon-
strated that feeding of fermented SBM (FSBM) can improve growth performance,
nutrient digestion, and physiological conditions in some fish species such as Atlantic
salmon (Refstie et al., 2005), rainbow trout (Matsunari et al., 2010), hybrid striped
bass (Rombensoa et al., 2013), Florida pompano, Trachinotus carolinus (Novriadi et
al., 2018), and large yellow croaker, Larimichthys crocea (Wang et al., 2019).
Pompano Trachinotus blochii (Lacepède, 1801), also known as silver pompano
or snubnose pompano, is a carnivorous marine fish species distributed mainly in
the Indo-Pacific region (Kapoor et al., 2002). It is one of the most preferred high
value species for mariculture, owing to its fast growth rate, good meat quality, and
high market demand (Othman, 2006). To date, no academic reports have described
the replacement of fish meal by SBM in pompano feed. Moreover, the inclusion of
FSBM in the diet is beneficial to growth performance and physiological conditions in
 Replacement of fish meal with soybean meals in pompano 577

some fish species; however, this diet has not yet been studied in pompano. Therefore,
the present study was conducted to examine the effects of the replacement of fish
meal by SBM and FSBM on the growth performance, nutrient apparent digestibility
(ADC), bile acid levels, and digestive enzyme activity in pompano fish.

Material and methods

Defatted and fermented soybean meals

Commercially available defatted SBM (dehulled SBM, crude protein [CP]
48%), fermented soybean meal 1 (FSBM1, CP 50%) and fermented soybean meal 2
(FSBM2, CP 51%) were used in this study. The FSBM1 was a commercially avail-
able product (product name Bio-S500) provided by Febecom J.S.C. (Phuc Khanh
industrial zone, Thaibinh, Vietnam). This meal was fermented by B. subtilis TH2.
The FSBM2 was produced through the method described by Nguyen et al. (2018).
Briefly, SBM (CP 48%) was steam cooked in an autoclave at 121°C for 20 min. After
cooling, the steamed SBM was inoculated by even spraying of a spore suspension of
B. subtilis B3 (105 [cfu]/ml, provided by Research Institute for Aquaculture No. 2,
Ho Chi Minh City, Vietnam). After thorough mixing, the inoculated SBM substrate
was incubated at 37°C for 72 h (pH 6.5, moisture 50%). After fermentation, the re-
sultant materials were dried in a vacuum drying oven at 60°C for 15 h. Finally, the
FSBM2 was ground to below 400 µm mesh size.

Experimental diets
Four isonitrogenous and isolipitic diets were formulated to replace 40% of fish
meal (260 g/kg diet) with SBM (400 g/kg diet), FSBM1 (390 g/kg diet) or FSBM2
(390 g/kg diet) (Table 1). The diets are denoted as follows: FMD (fish meal-based
diet), SBMD (fish meal replaced by SBM diet), FSBM1D (fish meal replaced by
FSBM1 diet), and FSBM2D (fish meal replaced by FSBM2 diet). Because me-
thionine is insufficient in SBM, this amino acid (5 g/kg diet) was supplemented
in SBMD, FSBM1D, and FSBM2D, according to previous studies (Nguyen et al.,
2013, 2017). Chromium oxide (5 g/kg diet) was added to all experimental diets as
an inert marker to estimate protein and lipid ADCs. All the powdered ingredients
were mixed manually for 5 min, and then transferred to a food mixer for another
15 min mixing. Chromium oxide was dissolved in distilled water and sprayed with
an atomizer on the mash during mixing, then pollock liver oil was added slowly to
the diet mixture. After the powdered ingredients were thoroughly mixed with pollock
liver oil, water was added to produce a stiff dough. The dough was then pelleted with
a laboratory pellet mill and stored at –30°C until use.

Fish husbandry
The experiment was carried out at The National Broodstock Center for Maricul-
ture Species, Research Institute for Aquaculture No. 1 (Catba, Haiphong, Vietnam).
Fingerling pompanos were acclimated to the experimental conditions for 2 weeks
578 H.P. Nguyen et al.

by FMD feeding before the start of the feeding trial. Thirty fish with an initial body
weight of 15 ± 0.3 g were allocated to each of the eight indoor polyvinyl chloride
tanks (500 L holding capacity), thus, resulting in two replicate tanks per dietary
treatment. The tanks were aerated and supplied with filtered seawater at a rate of
4 L/min. For 8 weeks, the fish were hand-fed the experimental diets to apparent
satiation twice daily (09:00 and 16:00). Dissolved oxygen and water temperature
were monitored daily and ranged between 5.8 ppm and 7.1 ppm, and between 25.7
to 28.8°C, respectively.

Table 1. Formulation and proximate composition of the experimental diets

Ingredients (g/kg) FMD SBMD FSBM1D FSBM2D
Fish meal 650 390 390 390
Defatted soybean meal 0 400 0 0
Fermented soybean meal 1 0 0 390 0
Fermented soybean meal 2 0 0 0 390
Corn gluten meal 70 40 40 40
Wheat flour 100 40 40 40
Pollock liver oil 60 90 90 90
Cellulose 90 5 15 15
Vitamin and mineral mixture 1
20 20 20 20
Methionine 0 5 5 5
CMC-Na 2
5 5 5 5
Chromium oxide 5 5 5 5
Proximate composition (dry matter basis, g/kg)
Crude protein 462 463 465 468
Crude lipid 128 125 124 127
Ash 128 103 107 105
1
Vitamin and mineral mixture (IU or mg/kg mixture): thiamine HNO3, 4.5; riboflavin, 7.5; pyridoxine HCl,
3.75; folic acid, 0.75; nicotinic acid, 22.5; calcium pantothenate, 7.5; biotin, 0.075; inositol, 15; choline chlo-
ride, 60; calcium L-ascorbate, 73.5; cyanocobalamin, 0.0225; vitamin A oil, 7.5; vitamin D3 oil, 18; vitamin E
(α-tocotrienol acetate), 27; menadione-NaHSO4, 3; FeSO4, 244.8; ZnSO4, 117.45; MnSO4, 106.38; CuSO4, 4.95;
MgSO4, 434.22; CoSO4, 4.56; calcium iodate, 0.525.
2
CMC-Na: sodium carboxymethyl cellulose.

Sampling
At the end of the feeding trial, fish were fasted for 48 h before sampling. All fish
were anesthetized with 400 ppm 2-phenoxyethanol and weighed individually. Blood
samples were collected with heparinized syringes from the caudal vein in five fish
in each tank, then centrifuged (10,000 rpm for 10 min) to obtain plasma. These fish
were also dissected to collect the gallbladder samples. The remaining fish was used
for fecal collection. For this purpose, fish were fed the same experimental diets, and
feces were collected by stripping at 4 h after feeding. After collection of sufficient
fecal matter for the determination of protein and lipid ADC, six fish from each tank
 Replacement of fish meal with soybean meals in pompano 579

were dissected at 3 h after feeding to collect the anterior intestinal digesta. The dis-
sected fish from each tank were divided into two groups (three fish each), and the
intestinal digesta from each group were pooled. The division of the intestinal tract
was based on the description by Murashita et al. (2008), and the anterior intestinal
digesta were collected from the entire straight region. All samples were maintained
at –30°C until analysis.

Analytical methods
Plasma constituents were analyzed with a commercial automatic analyzer (Ar-
chitect c16000, Abbott, Illinois, USA). Bile acids in freeze-dried intestinal digesta
were extracted with 90% ethanol, followed by chloroformmethanol (1:1, v/v), ac-
cording to the method described by Setchell et al. (1983). The extract from the di-
gesta and bile juice diluted with distilled water at ratio of 1:1200 were used for total
bile acid level quantification with a commercial assay kit (MAK309, Sigma-Aldrich,
St. Louis, MO, USA). Lipase and trypsin in freeze-dried intestinal digesta were ex-
tracted by homogenization into four and eight volumes (v/w) of cold distilled water,
respectively. The homogenates were then centrifuged at 20,000 rpm for 15 min. The
supernatant was further diluted 10-fold with cold distilled water. Lipase and trypsin
activities were measured with the method described by Murashita et al. (2008).
Briefly, lipase activity was measured as follows: a total of 150 μl of enzyme extract
was incubated with 0.4 mM p-nitrophenyl myristate (Sigma-Aldrich, St. Louis, MO,
USA) in 24 mM ammonium bicarbonate, 7.5 mM sodium deoxycholate, and 0.5%
Triton X-100, pH 8.5 (total volume: 1.5 ml). Lipase catalytic activity was determined
by measurement of the rate of p-nitrophenol (pNP) production at its optimal reac-
tion temperature (37°C). The increase in absorbance at 405 nm was recorded every
minute for 5 min. Reaction rates were calculated in units (U), such that 1 U was de-
fined as 1 μmol of pNP released in 1 min. Trypsin activity was measured as follows:
54 milligrams of N-benzoyl-L-arginine-p-nitroanilide (L-BAPA, Sigma-Aldrich, St.
Louis, MO, USA) was dissolved in 1 ml of dimethyl sulfoxide, and the volume was
adjusted to 100 ml with Tris buffer (0.1 M, pH 11, containing 20 mM CaCl2). The
reaction mixture consisted of 1.6 ml Tris buffer (0.1 M, pH 11, containing 20 mM
CaCl2), 1 mL L-BAPA, and 100 μl enzyme extract. Trypsin catalytic activity was
determined by measurement of the rate of p-nitroaniline (pNA) production at 50°C.
The increase in absorbance at 405 nm was recorded every minute for 5 min. Reaction
rates were calculated in U, such that 1 U was defined as 1 μmol of pNA released in
1 min. The proximate compositions of the experimental diets, the whole body, feces,
and the digestibility marker were analyzed according to the Association of Official
Analytical Chemists standard methods (AOAC, 2005). The ADC (%) was calculated
as 100 × [1 − (Idiet/Ifeces × Nfeces/Ndiet)], where Idiet and Ifeces represent the concentrations
of inert marker (chromium oxide) in the diet and feces, and Ndiet and Nfeces represent
the concentrations of nutrients in the diet and feces, respectively.

Statistical analysis
Data were analyzed with multivariate analysis of variance (MANOVA) to obtain
precise results for a small number of replicates (two) per experimental group. Pil-
580 H.P. Nguyen et al.

lai’s, Hotelling’s, and Roy’s tests of significance were applied. Differences between
means were assessed with the Tukey-Kramer test, and the significance threshold was
a 5% level of probability.

Results

Growth performance and feed utilization

The growth performance and feed utilization of pompano fed the experimental
diets are shown in Table 2. The final body weight (FBW), weight gain (WG), and
specific growth rate (SGR) of fish fed SBMD and FSBM1D were significantly lower
than those of fish fed FMD (P<0.05). There were no significant growth differences
between the FSBM2D and FMD experimental groups (P>0.05). The experimental
diets did not alter feed intake among the treatments. The feed conversion ratio (FCR)
was significantly higher in fish fed SBMD and FSBM1D rather than FMD (P<0.05),
whereas there was no significant difference in FCR between FMD and FSBM2D
(P>0.05).

Table 2. Growth performance and feed utilization of pompano fed the experimental diets1
Dietary groups
Parameters
FMD SBMD FSBM1D FSBM2D
Initial BW2 (g) 15.3±0.3 15.5±0.4 15.2±0.3 15.3±0.6
Final BW (g) 94.2±3.5 c 76.6±4.2 a 82.5±3.3 ab 89.7±2.6 bc
Weight gain3 (%) 515.6±11.7 d 394.0±13.9 a 442.7±11.3 b 486.4±5.0 cd
SGR (% BW/day)
4
3.25±0.03 c 2.85±0.05 a 3.02±0.04 ab 3.16±0.02 bc
FI (% BW/day)
5
4.57±0.10 4.82±0.12 4.75±0.09 4.66±0.18
FCR 6
1.75±0.03 a 2.04±0.03 c 1.93±0.02 bc 1.84±0.08 ab

1
Values are means ± standard deviations of two replicates. Values of each parameter in the same row with
different letters are significantly different (P<0.05).
2
Body weight (BW).
3
Weight gain = 100 × (final mean BW − initial mean BW)/initial mean BW.
4
Specific growth rate (SGR) = 100 × (ln final mean BW − ln initial mean BW)/feeding days.
5
Feed intake (FI) = 100 × total g dry feed intake/[(total initial BW + total final BW)/2]/feeding days.
6
Feed conversion ratio (FCR) = total g dry feed intake/(total final BW − initial total BW).

Plasma constituents
As shown in Table 3, the test diets did not significantly affect plasma total pro-
tein, glucose, and triglyceride levels in experimental fish (P>0.05). The total choles-
terol in fish fed SBMD was significantly lower than that in fish fed FMD (P<0.05).
There were no significant differences in total cholesterol among the FMD, FSBM1D
and FSBM2D experimental groups (P>0.05).
 Replacement of fish meal with soybean meals in pompano 581

Table 3. Plasma constituents of pompano fed the experimental diets1

Dietary groups
Parameters
FMD SBMD FSBM1D FSBM2D
Total protein (g/dl) 4.7±0.4 4.3±0.3 4.8±0.5 4.5±0.3
Glucose (mg/dl) 137.6±10.4 134.5±11.2 139.8±8.1 141.7±14.6
Total cholesterol (mg/dl) 275.2±22.4 b 206.0±17.5 a 231.6±20.0 ab 248.7±16.7 ab
Triglyceride (mg/dl) 179.3±16.2 165.4±12.4 171.3±18.2 177.1±14.3
1
Values are means ± standard deviations (n = 10). Values of each parameter in the same row with different
letters are significantly different (P<0.05).

Proximate composition of the whole body

The proximate composition of the whole body is presented in Table 4. The exper-
imental diets did not significantly alter the moisture, crude protein, and ash content
in fish (P>0.05). Crude lipids were significantly lower in fish fed SBMD rather than
FMD (P<0.05), whereas no significant differences were observed among the FMD,
FSBM1D and DSBM2D experimental groups (P >0.05).

Table 4. Proximate composition of the whole body of pompano fed the experimental diets (%)1
Dietary groups
Parameters
FMD SBMD FSBM1D FSBM2D
Moisture 64.8±1.3 65.7±0.9 65.4±1.1 65.1±0.8
Crude protein 15.5±0.7 14.8±1.2 15.2±0.7 15.4±0.5
Crude lipid 14.8±0.6 b 12.4±0.9 a 13.5±1.1 ab 14.3±0.7 ab
Ash 2.6±0.2 2.1±0.5 2.4±0.3 2.2±0.4
Values are means ± standard deviations (n = 10). Values of each parameter in the same row with different
1

letters are significantly different (P<0.05).

Table 5. Total bile acid level and digestive enzyme activity of pompano fed the experimental diets1
Dietary groups
Parameters
FMD SBMD FSBM1D FSBM2D
Total bile acid level
Gallbladder (mmol/l) 265.2±15.5 b 208.4±10.2 a 223.8±12.1 a 230.1±17.2 ab
Anterior intestine (μmol/g) 128.5±9.6 b 89.7±11.3 a 103.4±13.7 ab 112.6±8.8 ab
Digestive enzyme activity
Lipase (U/mg) 4.82±0.87 b 3.12±0.71 a 3.76±0.54 ab 4.22±1.01 ab
Trypsin (U/mg) 0.71±0.10 0.52±0.05 0.67±0.08 0.64±0.11
Values are means ± standard deviations (gallbladder, n = 10; intestinal digesta, n = 4). Values of each pa-
1

rameter in the same row with different letters are significantly different (P<0.05).

Total bile acid level and digestive enzyme activity

As presented in Table 5, the total bile acid levels in the gallbladder were signifi-
cantly lower in SBMD- and FSBM1D-fed fish than in FMD-fed fish (P<0.05); how-
ever, there was no significant difference between the FMD and FSBM2D experimen-
582 H.P. Nguyen et al.

tal groups (P>0.05). The total bile acid level in the anterior intestine was significantly
lower in the SBMD experimental group than the FMD experimental group (P<0.05),
whereas no significant differences were observed among the FMD, FSBM1D, and
FSBM2D experimental groups (P>0.05).
Lipase activity was significantly lower in fish fed SBMD than FMD (P<0.05).
There were no significant differences in lipase activity among fish fed FMD, FS-
BM1D, and FSBM2D (P>0.05). Trypsin activity was not significantly affected by
the experimental diets (P >0.05).

Protein and lipid ADC

As shown in Figure 1, protein ADC was not affected by the experimental diets.
In contrast, lipid ADC was significantly lower in fish fed SBMD and FSBM1D than
FMD (P<0.05). No significant difference in lipid ADC was found between the FMD
and FSBM2D experimental groups.

Figure 1. Apparent digestibility of lipid and protein in pompano fed the experimental diets. Values are
means and standard deviations of two replicates. Bars with different letters are significantly different
(P<0.05)

Discussion

In the present study, the FBW, WG, and SGR of fish fed SBMD were signifi-
cantly lower than those of fish fed FMD, whereas the FCR was significantly higher
for fish fed SBMD compared to FMD. These results indicate that replacement of
fish meal by SBM at a ratio of 40% in the diet impairs growth performance and
feed utilization of pompano fish. ANFs in SBM have been reported to negatively
affect growth performance, feed utilization, and physiological conditions in aquatic
animals, especially in carnivorous fish species. Soya antigens such as glycinin and
 Replacement of fish meal with soybean meals in pompano 583

β-conglycinin decrease digestive enzyme activity, and growth and feed performance
(Zhang et al., 2013; Li et al., 2017 a, b). High molecular weight fractions of soybean
protein decrease bile acid levels by interfering with bile acid reabsorption from the
intestine (Nguyen et al., 2011 a). Alcohol-soluble components in SBM, such as oli-
gosaccharides, saponins, and lectins, have been found to prevent the secretion of bile
acids and pancreatic lipases into the intestine, thereby resulting in poor dietary lipid
digestibility (Nguyen et al., 2011 b, 2017). In addition, alcohol-soluble molecules
from SBM, especially saponins and lectins, have been suggested to induce intestinal
morphological changes (Iwashita et al., 2009; Sorensen et al., 2011; Wang et al.,
2017). Therefore, the poor growth and feed performance of pompano fed SBMD in
the present study may be attributable to ANFs in SBM. Moreover, fish fed SBMD
showed significantly lower bile acid levels, lipase activity, and lipid ADC than those
fed FMD. These results suggest that poor dietary lipid digestion and absorption in
fish fed SBMD might be a factor responsible for growth retardation.
Fermentation has been suggested to be an effective method to reduce ANFs and
to improve the nutritional value of SBM. Refstie et al. (2005) have reported that
fermentation decreases the amount of ANFs, such as oligosaccharides, soy antigens,
and trypsin inhibitors, in SBM. Fermentation can also break down glycinin and
β-conglycinin, the major antigenic proteins in soybean, into smaller peptides and
this enhances growth performance and nutrient digestibility (Hong et al., 2004; Feng
et al., 2007; Shiu et al., 2015; Zhuo et al., 2016). In addition, the biotransformation
of substances and the production of secondary metabolites, such as antimicrobial
peptides, polyketides, isoflavone and saponin derivatives, during fermentation are
believed to have the potential to improve soybean nutritional properties and pro-
vide important health benefits (Lee et al., 2014 a, b; Harwood et al., 2018). Hence,
the better growth performance and feed utilization observed in fish fed FSBM2D
rather than SBMD in the present study may be due to the reduction of ANFs and the
improvement in the nutritional quality of SBM through the fermentation process.
Fermentation of SBM with B. subtilis has been shown to improve growth perfor-
mance and feed utilization in some fish species, such as rainbow trout (Matsunari
et al., 2010; Choi et al., 2020), yellowtail (Nguyen et al., 2013), orange-spotted
grouper Epinephelus coioides (Shiu et al., 2015), Florida pompano (Novriadi et al.,
2018), and largemouth bass Micropterus salmoides (He et al., 2020). In the current
study, FSBM1 was fermented by B. subtilis TH2, whereas FSBM2 was fermented by
B. subtilis B3. Fish fed FSBM1D showed a slightly higher growth and feed perfor-
mance than those fed SBMD; however, this diet did not restore the performance of
fish fed FMD. In contrast, fish fed FSBM2D showed growth and feed performance
comparable to those of fish fed FMD. Improvements in the growth performance and
feed utilization of fish fed FSBM are attained to the microorganisms used for fer-
mentation and fermentation conditions (Yamamoto et al., 2010; Nguyen et al., 2013;
Choi et al., 2020), the better performance of fish fed FSBM2D rather than FSBM1D
may be attributable to different fermentation processes and different bacterial strains.
The present findings suggest that fermentation of SBM with B. subtilis B3 may be
effective for enhancing growth performance and feed utilization of pompano fed
a soybean protein-based diet.
584 H.P. Nguyen et al.

Poor lipid digestion has been reported in Atlantic salmon (Olli and Krogdahl,
1995), rainbow trout (Romarheim et al., 2008), and yellowtail (Nguyen et al., 2011 a,
2017) fed SBM-based diets. In the current study, fish fed SBMD showed significant-
ly lower lipid ADC and whole body lipid content than those fed FMD. In contrast,
the lipid ADC and whole body lipid content of fish fed FSBM2D were comparable
to those of fish fed FMD. These results suggest that SBM impairs lipid digestion in
pompano fish and that the use of SBM fermented by B. subtilis B3 can improve lipid
digestion of the fish fed a soybean protein-based diet. Bile acids are important for
lipid digestion and absorption. They are essential not only for emulsification of lipids
and micelle formation but also for lipase activation (Gjellesvik et al., 1989; Tuch-
weber et al., 1996). In the present study, fish fed SBMD had significantly lower total
bile acid levels and lipase activity than fish fed FMD. However, these parameters
were elevated in fish fed FSBM2D, reaching values comparable to those in fish fed
FMD. These observations, together with the lipid ADC results, suggest that the better
lipid digestion of fish fed FSBM2D rather than SBMD may be due to increased bile
acid levels and lipase activity. In a previous study, high molecular weight fractions
of soybean protein have been found to decrease bile acid levels in the gallbladder
by interfering with bile acid reabsorption from the intestine (Nguyen et al., 2011 a).
In addition, alcohol-soluble components in SBM have been reported to inhibit the
secretion of bile acids and digestive enzymes into the intestine (Nguyen et al., 2011
b, 2017). Thus, in the current study, the reduction of such ANFs through the fermen-
tation process might be a reason for the increased bile acid levels and lipase activity
observed in fish fed FSBM2D.
In conclusion, the results of this study indicate that SBM decreases bile acid
levels, lipase activity, lipid ADC, and growth performance in pompano fish. These
parameters are improved by the inclusion of B. subtilis B3 fermented SBM in the
diet, thus, suggesting that fermentation of SBM with B. subtilis B3 may be an effec-
tive way to improve the digestive physiological conditions and growth performance
of pompano fed an SBM-based diet.

Acknowledgements
This study was funded by the Vietnam National Foundation for Science and
Technology Development (NAFOSTED) under grant number 106.05-2017.21. We
express special thanks to Prof. Dr. Masumoto Toshiro (Faculty of Agriculture and
Marine Sciences, Kochi University, Japan) for critical support and valuable sugges-
tions. We are grateful to staff at The National Broodstock Center for Mariculture
Species, Research Institute for Aquaculture No. 1 for their support during experi-
ments. We also thank A.P. Nguyen (Faculty of Biology, Hanoi National University
of Education, Vietnam) for technical assistance during fish sampling. There are no
conflicts of interest in this study.

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Received: 17 V 2020
Accepted: 29 VII 2020
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