Optimum fish meal replacement by soy protein concentrate from soymilk and phytase supplementation in diet of red sea bream, Pagrus major

Accepted Manuscript Optimum fish meal replacement by soy protein concentrate from soymilk and phytase supplementation in diets of red sea bream, Pagrus major

Amal Biswas, Hideo Araki, Tetsuo Sakata, Toshihiro Nakamori, Kenji Takii PII: DOI: Reference:

S0044-8486(19)30143-7 https://doi.org/10.1016/j.aquaculture.2019.03.023 AQUA 633981

To appear in:

aquaculture

Received date: Revised date: Accepted date:

18 January 2019 12 March 2019 12 March 2019

Please cite this article as: A. Biswas, H. Araki, T. Sakata, et al., Optimum fish meal replacement by soy protein concentrate from soymilk and phytase supplementation in diets of red sea bream, Pagrus major, aquaculture, https://doi.org/10.1016/ j.aquaculture.2019.03.023

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Optimum fish meal replacement by soy protein concentrate from soymilk and phytase supplementation in diets of red sea bream, Pagrus major

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Amal Biswasa*, Hideo Arakib, Tetsuo Sakatac, Toshihiro Nakamoric, and Kenji

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Aquaculture Research Institute, Kindai University, Uragami, Wakayama 649-5145, Japan

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Research Institute for Creating the Future, Fuji Oil Holdings Inc., Tsukubamirai, Ibaraki 300-

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Takiia

Fuji Oil Holdings Inc., Tsukubamirai, Ibaraki 300-2497, Japan

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2497, Japan

*Corresponding author: Tel: +81-735-58-0116 Fax: +81-735-58-1246 E-mail: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT

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Two trials were carried out to determine the optimal replacement level of fish meal (FM) by soy protein concentrate (SPC) derived from soymilk without supplementation of indispensable amino acids (IAAs) or palatability enhancers and whether phytase supplementation can help to replace more SPC in diet of juvenile red sea bream, Pagrus major. In Trial 1, five isoenergetic diets were formulated: 67% FM as protein source in the control diet (C), and FM was replaced by 60 (SPC60), 70 (SPC70), 80 (SPC80) and 100% (SPC100) by SPC. In Trial 2, diets C and SPC80 were the same as in Trial 1, and phytase was supplemented in SPC80 at 1000 (P1), 2000 (P2), 3000 (P3) and 4000 (P4) FTU/kg diet. Thirty (ca. 23 g) and 20 fish (ca. 21 g) were randomly distributed into each fifteen and eighteen 300 L indoor tanks in Trial 1 and 2, respectively. Fish in both trials were fed two times daily until apparent satiation for 10 weeks. At the end of both trials, feces were collected after feeding with chromic oxide (Cr2O3) mixed diets. In Trial 1, there were no significant differences in final mean weight, specific growth rate (SGR), daily feeding rate (DFR), feed efficiency (FE), condition factor (CF), and retention efficiency of protein, lipid and energy until 70% (SPC70) FM replacement by SPC (P > .05). However, 80% FM replacement significantly reduced final mean weight, FE and lipid retention efficiency compared with group C (P < 0.05). There were no significant differences in plasma constituents, except total cholesterol level. In Trial 2, phytase was supplemented in SPC80 to determine whether it helps to stimulate growth up to the level of the control group. Fish fed SPC80 once again displayed significantly lower final mean weight, SGR, FE, phosphorus (P) digestibility, and retention efficiency of protein, lipid, energy and P compared with control group (P > .05). Final mean weight, FE, P digestibility, and retention efficiency of protein, lipid and P were significantly improved in fish fed diet P2 (phytase at 2000 FTU/kg diet) compared with those fed diet SPC80. However, phytase supplementation did not stimulate the growth up to the level of group C. The results demonstrated in juvenile red sea bream that 70% of FM can be replaced by SPC derived from soymilk without supplementation of IAAs and palatability enhancers. Keywords: Red sea bream, soymilk protein, fish meal, growth, phytase

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1. Introduction

In recent decades, the increasing demand and stagnant or decreasing supply of fish meal

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(FM) has resulted in higher prices, which has prompted immediate action in the search for alternative protein sources. Among the terrestrial animal and plant protein sources available,

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soybean meal (SM) has been identified as a strong preference for FM replacement, mainly due to its compatible nutritional composition, relatively well-balanced amino acid (AA) profile,

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widespread availability, and low cost (Gatlin et al., 2007; Storebakken et al., 1998). However,

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there are mixed results on its utility in different fish species due to several limitations including anti-nutritional factors (ANFs), low levels of methionine and crude protein contents, and

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adverse effects on the intestinal integrity of some carnivorous species (Gatlin et al., 2007; Yamamoto et al., 2008; Krogdahl et al., 2010). While comparatively higher (>75%) or total

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replacement of FM by SM was reported in the diet of Senegalese sole, Solea senegalensis (Aragão et al., 2003); cobia, Rachycentron canadum (Salze et al., 2010); rainbow trout,

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Oncorhynchus mykiss (Kaushik et al., 1995); it was found that only 20-50% could be replaced in gilthead sea bream, Sparus aurata (Kissil et al., 2000); Japanese flounder, Paralichthys olivaceus (Deng et al., 2006); turbot, Scophthalmus masimus (Day and González, 2000); Korean rockfish, Sebastes schlegeli (Lim et al., 2004), spotted rose snapper, Lutjanus guttatus (Silva-Carrillo et al., 2012) and hybrid grouper (Faudzi et al., 2018).

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The utilization of further processed SM, such as defatted (Tantikitti et al., 2005), dehulled (Choi et al., 2004), heated (Peres et al., 2003), solvent-extracted (Boonyaratpalin et al., 1998), fermented (Refstie et al., 2005; Yamamoto et al., 2010) and gamma-irradiated (Zhang et al.,

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2014) in fish diets has been investigated in the above-mentioned studies. In juvenile red sea bream Pagrus major, which is one of the most important marine species in Japan, while 39%

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of FM replacement by conventionally processed SM without indispensable amino acids (IAAs) supplementation significantly reduced growth (Biswas et al., 2007a), soy protein concentrate

However, when SPC, soy protein isolate and soy peptides

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50% FM (Takagi et al., 1999).

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(SPC) produced through solvent extraction of SM in combination with krill meal could replace

derived from defatted soymilk were tested (Biswas et al., 2017), they could replace 57-77% of

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FM without compromising the growth performance of red sea bream. The feed formulation was intended to be simple in an effort to avoid IAA supplementation or the inclusion of

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palatability enhancers, except for taurine. Although enzyme-treated soy protein isolate could replace 77% of FM, it is not cost-effective. Among the soy products derived from soymilk used

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in the referenced study, SPC is less expensive than other products. In previous reporting (Biswas et al., 2017), a fixed amount (30%) of different forms of soy protein from soymilk were incorporated in the diet, which was equivalent to 57% of FM replacement by SPC. In an effort to build upon these results, it is necessary to determine the maximum replacement level of FM by SPC.

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In red sea bream, it was found that phytase supplementation significantly improved the growth performance when conventionally processed SM was used to replace FM (Biswas et al. 2007a). The positive effect of phytase supplementation has also been reported in other species

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(Forster et al., 1999; Cheng and Hardy, 2003; Yoo et al., 2005; Sajjadi and Carter, 2004; Hien et al., 2015). However, the inclusion of phytase did not improve the growth performance in

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some studies (Forster et al., 1999; Vielma et al., 2000; Sajjadi and Carter, 2004). It may be related to the difference in content of ANFs in ingredients and species-specific ability to utilize

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the plant protein source. Therefore, this study aimed to determine the optimum replacement

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level of FM by SPC from soymilk without supplementation of IAAs or palatability enhancers,

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and whether phytase supplementation can further improve the utilization of SPC.

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2. Materials and methods

Two trials were conducted at the Aquaculture Research Institute, Uragami Station of Kindai

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University, Japan. In Trial 1, SPC derived from defatted soymilk was evaluated on its maximum inclusion level in the diet. In Trial 2, phytase was supplemented at different dosages in diet SPC80, which produced inferior results compared with the control group in Trial 1. Phytase supplementation rates were analyzed to determine if it could improve the growth performance to a level comparable to the control group.

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2.1 Trial 1

2.1.1 Experimental diets, fish and husbandry

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SPC (protein, ca. 63%) was provided by Fuji Oil Holdings Inc. (Osaka, Japan). All other dietary components were obtained commercially, except for FM (protein, ca. 67%), which was

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provided by Chubu Feed Co. Ltd. (Nagoya, Japan). Five experimental diets were formulated to be nearly isonitrogenous, isolipidic and isocaloric according to the formula given in Table 1.

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In control diet (C), FM was used as the sole protein source. As in a preliminary experiment

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with different forms of soy protein from soymilk (Biswas et al., 2017), SPC could comfortably replace 57% of FM, and feed efficiency (FE) was slightly better than the control group.

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Therefore, four other diets were prepared by substituting 60, 70, 80 and 100% of FM using a combination of SPC and corn gluten (approximately 4:1), and subsequently referred to as

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SPC60, SPC70, SPC80 and SPC100, respectively. Corn gluten (protein, ca. 63%) was included in SPC diets to compensate some amino acids. Similar to the previous study by Biswas et al.

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(2017), the feed formulation did not include IAA or palatability enhancement supplementation, except for taurine. All ingredients provided in the formula were mixed well before adding water. After adding water, the dough was pelletized using a laboratory pellet machine with an appropriate diameter, freeze-dried at -80°C and stored at -20°C until used. Approximately seven hundred juveniles red sea bream were obtained from the fish rearing facility of Kindai University, Japan and stocked into a 3000 L tank for acclimation. Fish were

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fed to apparent satiation for a two-week acclimation period with a commercial diet (protein 47.7%, lipid 10.7%, Marubeni Nisshin Feed Co. Ltd., Tokyo, Japan), twice a day at 09:00 and 15:00.

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Fish were starved for 24 h after conditioning for two weeks, and 30 fish (mean body weight ca. 23 g) were randomly distributed into fifteen-300 L (100 × 50 × 60 cm) indoor tanks and

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set in triplicate for each treatment. Fish were fed to apparent satiation with the experimental diets twice per day at 09:00 and 15:00, 6 days per week, for 10 weeks. The photoperiod for all

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treatments was maintained at 12-h light (07:00~19:00):12-h dark. Tanks were supplied with

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filtered seawater at a rate of 7 L/min and aerated to maintain dissolved oxygen levels near 100% saturation. The mean water temperature and dissolved oxygen during the rearing trial

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were 26.3±0.8°C and 7.5±1.2 mg/L, respectively. Bottom cleaning was carried out once per day and dead fish were collected and weighed, if any mortalities were present. Fish were

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weighed in pool biweekly after starving for 24 h.

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2.1.2 Fish sampling

At the beginning of the trial, a pooled sample of 30 fish was stored in a freezer (-80°C) for whole-body proximate analyses. At the end of the trial, fish were fasted for 24 h before being anesthetized using 200 ppm 2-phenoxyethanol (Wako Pure Chemical Industries Ltd. Osaka, Japan) and all survived fish from each tank were counted and weighed in pool. Three fish were randomly selected from each of the triplicate tanks for each treatment at the end of rearing trial,

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and approximately 1 ml of blood was collected to separate plasma after centrifuging at 2000 g for 15 minutes at 4°C. The plasma was stored at -80°C until analysis. After blood sampling, an additional 10 fish were randomly selected from each of triplicate tanks for each treatment and

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pooled to use in the digestibility experiment. Additionally, 3 fish from each tank were dissected to determine relative organ weight, and the remaining fish from each tank were frozen at -80°C

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for whole body proximate analysis.

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2.1.3 Feces collection

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Since an earlier study demonstrated that a single tank for each treatment can be used to get reliable measurements for apparent digestibility coefficient (ADC) of nutrients and energy

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(Biswas et al., 2007b), total 30 fish from each treatment for digestibility experiment were transferred to a single 350 L tank with a fecal settling column. Fish were fed with the same

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experimental diets as in the growth trial, with the inclusion of 0.5% chromic oxide (Cr2O3) as an inert marker. All rearing conditions were similar to growth trial and feces were collected

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after 2 weeks acclimation to the new tank. Feces were collected at 08:30 prior to the first feeding for 3 consecutive days at the 3rd week for analyses. About 1.5 h after the last feeding (15:00) of previous day, tank walls and fecal collectors were thoroughly cleaned to prevent uneaten pellets from settling at the tank bottom or remaining in the fecal settling column to avoid feces contamination. After collecting fecal samples from the settling column, excess water was separated immediately by centrifugation (2000 g for 30 min at 4°C). All collected

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feces were stored at -80°C and freeze-dried prior to analysis. At the end of the digestibility trial, fish were starved for 24 h, and body length and weights were measured to clarify if growth

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performance displayed a consistent pattern to that of the growth trial.

2.2.1 Experimental diets, test fish and husbandry

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2.2 Trial 2

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Since phytase supplementation significantly improved the growth performance when

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conventionally processed SM was used to replace FM (Biswas et al. 2007a) in red sea bream, the aim of this trial was to investigate whether the significantly lower growth performance from

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SPC80 in Trial 1 could be increased up to the performance level of the control group. The sources of all ingredients were the same as in Trial 1. Dietary formula for control (C) and

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SPC80 was the same as in Trial 1 (Table 2). Apart from diets C and SPC80, four other isonitrogenous, isolipidic and isocaloric diets were formulated by supplementing SPC80 with

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phytase (BASF, Tokyo, Japan) at 1000, 2000, 3000 and 4000 phytase activity units (FTU) per kg diet (0.02, 0.04, 0.06 and 0.08 g/100 g diet, respectively), and referred to as P1, P2, P3 and P4, respectively. One FTU is defined as the amount of enzyme that generates 1 µmol of inorganic phosphorus (P) per min from an excess of sodium phytate at pH 5.5 and 37°C. The diets were pelletized, freeze dried and stored similarly to Trial 1. Five hundred red sea bream juveniles were obtained from the fish rearing facility of Kindai

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University, Japan and stocked into a 3000 L tank for a two-week acclimation period using the same rearing conditions and feeding protocol as in Trial 1. Subsequent to acclimation, fish were starved for 24 h, and 20 fish (mean body weight of

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approximately 21 g) were randomly distributed into eighteen-300 L (100 × 50 × 60 cm) indoor tanks in triplicate, for each dietary treatment. Feeding protocol, rearing period and other

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husbandry practices were consistent with Trial 1. However, the mean water temperature and

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dissolved oxygen during the rearing trial were 27.0±1.2°C and 7.4±0.9 mg/L, respectively.

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2.2.2 Fish sampling

For whole body proximate analysis, a pooled sample of 20 fish was stored in a freezer (-

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80°C) at the beginning of the trial. At the end of the trial, fish were fasted, anesthetized and weighed in pool consistent with Trial 1. However, 2 fish for blood collection, 2 fish for relative

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organ weight determination and an additional 8 fish were randomly selected from each of the triplicate tanks to use in the digestibility experiment. The remaining fish from each tank were

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frozen at -80°C for whole body proximate analysis.

2.2.3 Feces collection A total of 24 fish from each treatment for digestibility experiment were transferred to a single 350 L tank with a fecal settling column and fed with the same experimental diets as in the growth trial, with the additional inclusion of 0.5% chromic oxide (Cr2O3) as an inert marker.

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After two weeks acclimation to the fecal collection column, all necessary steps regarding feces collection were carried out as in Trial 1.

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2.3 Biochemical analyses and growth parameters calculation

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The diets, initial and final whole body, and feces were analyzed for moisture, crude protein, lipid and ash, in triplicate, using standard methods (AOAC, 1995). Dietary crude sugar was

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measured using the phenol-sulfuric acid method (Hodge and Hofreiter, 1962). The gross energy

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contents of fish, diets and feces were determined directly using an automated oxygen bomb calorimeter (IKA-Werke GmbH & Col KG, Germany). P content of the diets, fish whole body

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and feces was determined using the ammonium-molybdate method described by Baginski et al. (1982) after the digestion of samples with nitric and perchloric acids. Chromic oxide in the

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diets and feces was determined by a wet-acid digestion method (Furukawa and Tsukahara, 1966). AAs concentration in the diets was analyzed using high performance liquid

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chromatography (HPLC, GL-Science Inc., Tokyo, Japan) as described by Biswas et al. (2017). Plasma constituents were determined by a commercial kit using Fuji Dry-chem (Fujifilm Company Ltd., Tokyo, Japan). The following formulae were used to calculate different growth parameters. Specific growth rate, SGR (%/day) = 100  (ln final weight – ln initial weight)/time (days). Daily feeding rate, DFR (g/100g fish/day) = 100  total feed intake / [(mean of initial and

ACCEPTED MANUSCRIPT final no of fish  mean of initial and final body weight)/rearing period] Feed efficiency, FE (%) = 100  [wet weight gain (g) / dry feed intake (g)] Condition factor, CF = 100  (W / L3), where, W = wet body weight (g) and L = body length

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(cm) Retention efficiency of protein, lipid or energy (%) = 100  [(final whole-body protein,

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lipid or energy – initial whole-body protein, lipid or energy)/total protein, lipid or energy intake]

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Viscerosomatic index, VSI (%) = 100  [wet weight of viscera and associated fat (g) / wet

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body weight (g)]

Hepatosomatic index, HSI (%) = 100  [wet weight of liver (g) / wet body weight (g)]

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Stomatosomatic index, SSI (%) = 100  [wet weight of stomach (g) / wet body weight (g)]

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Intestinosomatic index, ISI (%) = 100  [wet weight of intestine (g) / wet body weight (g)]

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ADC of protein, energy or P (%) = 100  [1 – {(dietary Cr2O3/fecal Cr2O3)  (fecal protein,

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energy or P /dietary protein, energy or P)}]

2.4 Statistical analyses

All statistical analyses were carried out using the SPSS program for Windows (v. 10.0). Data were expressed as the mean  S.D. of triplicate samples. The means within each treatment and among different treatments were compared using an ANOVA followed by Tukey’s test of

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multiple comparison. Probabilities of P < 0.05 were considered significant. For statistical analysis of digestibility, three variables from 3 consecutive days of sampling were classified as

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one data set for each treatment.

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3. Results

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3.1 Trial 1

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Since no additional IAAs were supplemented in SPC based diets, histidine, lysine, methionine, threonine and valine contents were lower than those of control diet. When IAAs

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index was calculated considering diet C as reference, it ranged from 83.7 to 88.2 in SPC-based diets (Table 3).

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The growth performance of Trial 1 is shown in Table 4. Although there was no significant difference in final mean body weight among diets C, SPC60 and SPC70, it was significantly

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lower in diets SPC80 and SPC100 (P > .05). A similar trend was observed in SGR, but there was no significant difference between fish fed diets SPC70 and SPC80. Survival and DFR showed a similar pattern, where fish fed with diet SPC100 represents significantly lower values than those of fed with other diets (P > .05). FE was significantly reduced in fish fed diets SPC80 and SPC100; however, there was no significant difference among diets C, SPC60 and SPC70. CF was decreased gradually with increasing level of SPC in diet, SPC100 being represented

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significantly the lowest value (P > .05). Fish fed with diet SPC100 showed significantly lower final whole-body moisture, protein, lipid and energy contents than those fed with other diets (P > .05, Table 5). Although there was

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no significant difference in protein digestibility among the treatments, it was decreased gradually with increasing FM replacement levels. Fish fed with diet SPC100 exhibited

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significantly lower protein and lipid retention efficiency than other diets (P > .05); however, energy retention efficiency in fish fed with both SPC80 and SPC100 was significantly lower

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than those fed with C and SPC60.

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The variation in relative organ weight and plasma constituents are shown in Table 6. VSI and HSI were significantly lower in fish fed with SPC100 than those of group C (P > .05).

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However, there were no significant differences in SSI and ISI among the treatments (P > .05). Although variations were found in some of the plasma parameters, there were no significant

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differences among the treatments except for that of total cholesterol, which showed significant

3.2 Trial 2

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decreasing trend with increasing FM replacement by SPC.

Final mean body weight, SGR and FE in fish fed with all SPC-based diets, irrespective of phytase supplementation, were significantly reduced compared with group C (P > .05, Table 7). However, phytase supplementation at 1000 and 2000 FTU/kg diet in P1 and P2 significantly

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improved final mean weight and FE compared with SPC80 (P > .05). In contrast, DFR was significantly higher in fish fed with all SPC-based diets compared with group C. There were no significant differences in survival and CF among the treatments (P > .05).

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Whole body proximate composition, ADC and retention efficiency are shown in Table 8. There were no significant differences in whole body proximate composition among the

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treatments, expect the lipid content. Although there was no significant difference in lipid content among C and all phytase supplemented groups, it was significantly reduced in fish fed

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with SPC80 compared with group C. While apparent digestibility of protein and energy were

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similar among the treatments, that of P was improved significantly in phytase supplemented groups as compared with diets C and SPC80. Retention efficiency of protein, lipid and energy

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were significantly higher in fish fed with diet C than other dietary groups (P > .05). Among phytase supplemented groups, P2 produced significantly higher protein and lipid retention

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efficiency than SPC80 group. P retention efficiency was significantly lower in fish fed with SPC80 than all other dietary groups (P > .05).

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Neither relative organ weight nor plasma constituents showed significant differences among the treatments, except plasma total cholesterol level (Table 9). Fish fed with SPC80 showed significantly lower total cholesterol content than that of group C (P > .05).

4. Discussion

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4.1 Trial 1

Although all growth parameters in SPC100 and the most in SPC80 were significantly lower,

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there were no significant differences when 70% of FM was replaced (SPC70) by SPC compared with the control group. In previous study, even 39% of FM replacement by conventionally

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processed SM produced significantly lower growth performance in juvenile red sea bream (initial mean weight ca. 24 g) without supplementation of IAAs and palatability enhancers

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(Biswas et al., 2007a). However, SPC produced through solvent extraction of SM to increase

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protein content could replace 50% FM in juvenile red sea bream (initial mean weight ca. 33.5 g) when combined with krill meal (Takagi et al., 1999). In this study, SPC derived from soymilk

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instead of SM and could replace 70% of FM in juvenile red sea bream without compromising the growth performance. ANFs and essential IAAs deficiency are the main disadvantages of

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plant-derived feedstuffs when used in aquafeeds as supplements to FM (Gatlin et al., 2007). Therefore, it is necessary to reduce or inactivate the ANFs in plant protein through appropriate

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processing to achieve optimal nutritional potential (Wilson and Poe, 1985; Van der Ingh et al., 1991). The SPC used in this study was purified using aqueous and alcoholic or acidic extraction followed by the heat treatment of soymilk, which reduced the trypsin inhibitor activity (TIA) from 76.2 TIU/mg sample in conventionally processed SM to 32.3 TIU/mg sample in SPC, as reported by Biswas et al. (2017). Although other ANFs were not analyzed except saponin, the lower TIA value and processing of SPC derived from soymilk could be some of the plausible

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reasons of greater utilization by juvenile red sea bream. Similarly, the utilization of further processed SM has been investigated in other species as mentioned earlier (Boonyaratpalin et al., 1998; Peres et al., 2003; Choi et al., 2004; Refstie et al., 2005; Tantikitti et al., 2005;

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Yamamoto et al., 2010; Zhang et al., 2014). For example, Zhang et al. (2014) demonstrated that dietary FM can be replaced only up to 25% when untreated SM was used, while the

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replacement level could be increased up to 50% using gamma-irradiated SM. Since the utilization capacity of alternative protein sources in red sea bream tends to increase with growth

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(Takagi et al., 1999, 2000), it is necessary to investigate if SPC used in this study can replace

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more or total replacement of FM at other growth stages. Interestingly, Kader et al. (2012) suggested that FM can be completely replaced using a combination of solvent-extracted

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dehulled SM, fish soluble (FS), krill meal (KM), squid meal (SQM) and highly unsaturated fatty acids (HUFA) in the diet of red sea bream. However, the present studies aim to develop a

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practical diet without supplementation of FS, KM, SQM and HUFA, which similar to FM are both expensive and are at risk for future availability.

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When developing diets containing a high level of plant protein sources to replace FM, palatability is a major concern with respect to maintaining feed intake (FI). In general, the impaired growth performance in carnivorous fish fed plant protein-based diets is caused by reduced feed palatability and FI due to unfavorable taste (Nagel et al., 2012). In juvenile red sea bream, significantly lower FI was observed when SM or SPC was used to replace FM (Biswas et al., 2007a; Kader et al., 2010). However, when FS, KM, SQM, and some deficient

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IAAs (lysine, methionine) were supplemented with plant protein-based diets, the FI was either equal to or significantly improved compared to the FM-based control group in juvenile red sea bream (Takagi et al., 1999, 2001; Kader et al., 2010, 2012). As mentioned earlier, the feed

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formulation utilized in this study was intended to be simple to avoid the need for supplementation of palatability enhancers or IAAs. Nevertheless, there were no significant

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differences in FI until 80% FM replacement compared with the control group. While similar or higher FI in plant protein-based diets is reported to be correlated with remarkable or

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significantly lower FE as compared to FM-based diets in juvenile red sea bream (Biswas et al.,

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2007a; Kader et al., 2010; Takagi et al., 1999), FE didn’t significantly decrease until 70% replacement of FM by SPC used in this study. Moreover, there were no significant differences

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in ADC of protein, and retention efficiency of protein, lipid and energy until 70% FM replacement by SPC. In a preliminary study, the equal growth performance, ADC and retention

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efficiency of protein and lipid between control and 57% FM replaced by SPC indicated that the supplementation of IAAs or palatability enhancers were not necessary when SPC from soymilk

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was used in the diet of juvenile red sea bream (Biswas et al., 2017). In addition, this study revealed that FM can be replaced even up to 70% by SPC derived from soymilk without further supplementation of IAAs or palatability enhancers. It may be difficult to compare the results from this study directly to those of others in juvenile red sea bream mentioned above due to some differences in fish size and husbandry methodology. However, it seems that SPC from soymilk may be more utilizable by this species as compared with conventionally processed SM

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or SPC from SM. Since the presence of ANFs and low digestibility of protein are some of the factors limiting FM replacement by plant protein-based diets (Francis et al., 2001; Gatlin et al., 2007), a remarkably lower TIA activity in SPC used here as compared with conventionally

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processed SM (Biswas et al., 2017) could be a reason for improved utilization through an increased digestibility and retention efficiency of protein and lipid. Takagi et al. (2001) reported

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that the supplementation of crystalline methionine and lysine can improve growth and feed utilization in juvenile red sea bream when FM was replaced by SPC from soybean meal.

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Although methionine and lysine contents in diets SPC60 and SPC70 were lower and the IAA

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index was about 88.2 compared to the control group, the similar growth performance in these two groups may suggest that the palatability, protein digestibility, methionine and lysine

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contents are not factors that negatively affect growth and feed utilization in juvenile red sea bream when SPC is derived from soymilk. In Japanese seabass, Zhang et al. (2014) found no

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significant changes in FI and digestibility from untreated SM-based diets when maintaining the same levels of digestible protein, methionine and lysine to that of control group, suggesting

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that palatability, digestibility and methionine content were not factors that influence inferior growth performance.

Among blood parameters, GOT (or aspartate aminotransferase, AST) and GPT (or alanine aminotransferase, ALT) are often used to evaluate liver function, as they are released into the blood if there is injury or damage to the liver cells of the fish (Lemaire et al., 1991). It is reported that these two protein metabolism enzymes increase when available IAAs are deficient

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(Cheng et al., 2010; Wang et al., 2016). Although Kader et al. (2010) found a significant increase in both parameters from an SPC-based diet in red sea bream juvenile, the lack of significant differences among the treatments in this study suggests that the liver may not be

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affected. The total cholesterol level showed a significant decreasing trend with increasing FM replacement, which is in agreement with the findings of other studies when red sea bream was

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fed with SPC-based diets (Takagi et al., 1999; Kader et al., 2010). However, the blood parameters, which serve as reliable indicators for the overall health and physiological condition

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of the fish, provided in this article are within the normal range for juvenile red sea bream as

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compared to those of previous findings (Aoki et al., 1998; Takagi et al., 1999, 2001; Kader et al., 2010, 2012; Uyan et al., 2007; Biswas et al., 2017). Therefore, the data from this study

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suggests that roughly 70% of FM can be replaced by SPC derived from soymilk without

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4.2 Trial 2

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compromising the growth performance and health status of juvenile red sea bream.

As discussed above, though SPC derived from soymilk successfully replaced 70% of FM without compromising the growth performance in Trial 1, final mean body weight, FE and energy retention efficiency were significantly reduced in the 80% replacement group (SPC80). However, there were no significant differences in DFR, survival rate, condition factor, protein digestibility and retention efficiency of protein and lipid between the control and 80%

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replacement groups. A previous study of juvenile red sea bream reported significant improvement of growth when phytase was supplemented in conventionally processed SMbased diet (Biswas et al., 2007a). Therefore, phytase was supplemented at different dosages in

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the 80% replacement diet (SPC80) to determine if the inferior growth parameters from Trial 1 could be improved up to the level of control group.

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When the growth performance between fish fed diets C and SPC80 was compared, a similar trend to that of Trial 1 was observed. Final mean weight, SGR and FE were significantly

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reduced in fish fed diet SPC80, similar to Trial 2. However, final mean weight and FE were

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significantly improved when phytase was supplemented at 1000 and 2000 FTU/kg diet as compared with those found in fish fed diet SPC80 without phytase supplementation. In other

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species, while improved growth performance was found when fed with either phytase supplemented diets (Jackson et al., 1996; Papatryphon et al., 1999) or phytase pretreated

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ingredients (Vielma et al., 2002), phytase could not improve the growth performance in other studies (Forster et al., 1999; Vielma et al., 2000; Masumoto et al., 2001; Sajjadi and Carter,

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2004; Yoo et al., 2005; Qiu and Davis, 2017). In this study, the growth performance was not improved at phytase dosages higher than 2000 FTU/kg diet, which is agreed with other study on juvenile red sea bream (Biswas et al., 2007a). Similar results were also observed in rainbow trout (Forster et al., 1999) and Korean rockfish (Yoo et al., 2005). However, the growth improvement through phytase supplementation did not reach the level of control group.

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Phytate, as an antinutritional factor found in plant-based protein, reduces the nutritional quality of the diet by affecting nutrients and minerals bioavailability through a negative effect on digestibility in different fish species (Rodehutscord and Pfeffer, 1995; Cao et al., 2007;

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Kumar et al., 2012). Phytases, a group of enzymes known as myoinositol-hexaphosphate phosphohydrolase, are ideal approaches to degrade phytate to sequentially produce myoinositol

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penta-, tetra-, tri-, di-, and monophosphates, which neutralizes the negative effects of phytate on protein and other nutrients in the diet of monogastric animals (Mitchell et al., 1997).

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Although statistically not significant, a remarkable increase in protein digestibility was

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observed in phytase supplemented SPC-based diets, which, in turn, significantly improved protein retention efficiency at phytase dosage 2000 FTU/kg diet compared with the non-

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supplemented group. This may be attributed to the fact that phytase can improve protein availability through the breakdown of phytin-protein complexes in the gut, and neutralize the

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negative influence of phytate on protein in the diet of monogastric animals (Mitchell et al., 1997; Liebert and Portz, 2005). Together with juvenile red sea bream (Biswas et al., 2007a),

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similar positive influence of phytase on protein utility was also reported in rainbow trout (Vielma et al., 2002), pangas catfish Pangasius pangasius (Debnath et al., 2005), rohu Labeo rohita (Baruah et al., 2004), yellow catfish Pelteobagrus fulvidraco (Zhu et al., 2014) and white shrimp Litopenaeus vannamei (Qiu and Davis, 2017). Similar to the previous study on juvenile red sea bream (Biswas et al., 2007a), the digestibility and retention efficiency of P showed more interesting results. Apparent P digestibility is considered to be the most sensitive criteria

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for evaluating the effect of phytase on P utilization. Phytase can help to prevent P pollution in the local environment by converting phytate-P into available P, which helps to improve P bioavailability and reduce inorganic P supplementation in the diet (Sugiura et al., 1999; Yoo et

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al., 2005). Although 80% of FM replacement significantly reduced ADC and retention efficiency of P in fish fed diet SPC80 in this study, both parameters increased significantly by

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12.6% in P4 and 54.0% in P2, respectively. This may be attributed to the activity of phytase which dephosphorylates phytic acid and phytate-P to increase the availability (Lanari et al.,

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1998; Storebakken et al., 1998). Similar results were reported in juvenile red sea bream (Biswas

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et al., 2007a), striped bass Morone saxatilis (Papatryphon et al., 1999; Papatryphon and Soares, 2001), Japanese flounder (Masumoto et al., 2001), Atlantic salmon Salmo salar L.

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(Storebakken et al., 1998; Sajjadi and Carter, 2004), Korean rockfish (Yoo et al., 2005) and rainbow trout (Sugiura et al., 2001; Vielma et al., 2002; Cheng and Hardy, 2003). The

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significant improvement of almost all growth parameters, digestibility and retention efficiency in fish fed P2 than that of SPC80 suggests that the optimal phytase supplementation level is

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2000 FTU/kg diet, which agrees with the previous study on juvenile red sea bream (Biswas et al., 2007a). Moreover, relatively lower P content but higher digestibility and retention efficiency in phytase supplemented groups suggest that those diets will be ecologically sustainable. Unfortunately, phytase supplementation in SPC80, irrespective of dosages, could not improve the growth performance up to the level of control group. Further studies are necessary to investigate on how the utility of SPC derived from soymilk can be improved to

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achieve greater FM replacement in the diet of red sea bream without supplementation of IAAs and palatability enhancers.

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5. Conclusion

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The results of these studies suggest that SPC derived from soymilk can replace as much as 70% of FM, which could be advantageous to the red sea bream aquaculture industry. Since the

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utilization of alternative protein sources increased with growth of fish (Takagi et al., 2001), it

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is necessary to investigate whether greater replacement can similarly maintain good growth at the grow-out stage. Although the improvement of P digestibility and retention efficiency

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through phytase supplementation at 2000 FTU/kg diet may ensure ecological benefits, the lack of growth improvement to the level of control group prompts further research. In previous

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reporting (Biswas et al., 2017), it was proposed that the reduced TIA activity in SPC from soymilk compared with conventionally processed SM might be one of the factors influencing

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higher utility of this product. Therefore, it is necessary to investigate whether further reduction of TIA content can support more replacement of FM in the diet of juvenile red sea beam.

Acknowledgements

The expenses of this study were partly defrayed by a project on ‘Sophistication of full-

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cycle culture of fish’ of Kindai University and the collaborative research fund from Fuji Oil Holdings Inc., Osaka, Japan.

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References AOAC, 1995. Official Methods of Analysis of AOAC International. vol I. Agricultural

SC

RI

Chemicals; Contaminants, Drugs, 16th edition. AOAC International, Arlington, VA, USA. 1298pp.

NU

Aoki, H., Furuichi, M., Viyakarn, V., Yamagata, Y., Watanabe, T., 1998. Feed protein

MA

ingredients for red sea bream. Suisanzoshoku 46, 121–127. Aragão, C., Conceicção, L.E.C., Dias, J., Marques, A.C., Gomes, E., Dinis, M.T., 2003. Soy

PT E

D

protein concentrate as a protein source for Senegalese sole (Solea senegalensis Kaup 1858) diets: effects on growth and amino acid metabolism of postlarvae. Aquacult. Res. 34,

CE

1443–1452.

Baginski, E.S., Slawa, S.M., Zak, B., 1982. Phosphate, inorganic. In: Baginski, E.S. (Ed.),

AC

Selected Methods of Clinical Chemistry. American Association Clinic Chemistry, Washington, DC, pp. 313-316. Baruah, K., Sahu, N.P., Pal, A.K., Debnath, D., 2004. Dietary phytase: An ideal approach for a cost effective and low-polluting aquafeed. NAGA, WorldFish Center Quarterly 27, 15– 19. Biswas, A.K., Kaku, H., Ji, S.C., Seoka, M., Takii, K., 2007a. Use of soybean meal and phytase

ACCEPTED MANUSCRIPT

for partial replacement of fish meal in the diet of red sea bream, Pagrus major. Aquaculture 267, 284–291. Biswas, A., Seoka, M., Takii, K., Kumai, H., 2007b. Comparison of the apparent digestibility

PT

coefficient among replicates and different stocking density in red sea bream, Pagrus major. Fish. Sci. 73, 19-26.

SC

RI

Biswas, A., Araki, H., Sakata, T., Nakamori, T., Kato, K., Takii, K., 2017. Fish meal replacement by soy protein from soymilk in the diets of red sea bream (Pagrus major).

NU

Aquacult. Nutr. 23, 1379-1389.

MA

Boonyaratpalin, M., Suraneiranat, P., Tunpibal, T., 1998. Replacement of fish meal with various types of soybean products in diets for the Asian seabass, Lates calcarifer.

PT E

D

Aquaculture 161, 67–78.

Cao, L., Wang, W., Yang, C., Yang, Y., Diana, J., Yakupitiyage, A., Luo, Z., Li, D., 2007.

CE

Application of microbial phytase in fish feed. Enzyme Microbial Technol. 40, 497–507. Cheng, Z.J., Hardy, R.W., 2003. Effects of extrusion and expelling processing, and microbial

AC

phytase supplementation on apparent digestibility coefficients of nutrients in full-fat soybeans for rainbow trout (Oncorhynchus mykiss). Aquaculture 218, 501–514. Cheng, Z., Ai, Q., Mai, K., Xu, W., Ma, H., Li, Y., Zhang, J., 2010. Effects of dietary canola meal on growth performance, digestion and metabolism of Japanese seabass, Lateolabrax japonicus. Aquaculture 305, 102–108. Choi, S.M., Wang, X., Park, G.J., Lim, S.R., Kim, K.W., Bai, S.C., Shin, I.S., 2004. Dietary

ACCEPTED MANUSCRIPT

dehulled soybean meal as a replacement for fish meal in fingerling andgrowing olive flounder Paralichthys olivaceus (Temminck et Schlegel). Aquacult. Res. 35, 410–418. Day, O.J., González, H.G.P., 2000. Soybean protein concentrate as a protein source for turbot

PT

Scophthalmus maxiums L. Aquacult. Nutr. 6, 221–228. Debnath, D., Pal, A.K., Sahu, N.P., Jain, K.K., Yengkokpam, S., Mukherjee, S.C., 2005. Effect

SC

RI

of dietary microbial phytase supplementation on growth and nutrient digestibility of Pangasius pangasius (Hamilton) fingerlings. Aquacult. Res. 36, 180–187.

NU

Deng, J., Mai, K., Ai, Q., Zhang, W., Wang, X., Xu, W., Liufu, Z., 2006. Effects of replacing

MA

FM with soy protein concentrate on feed intake and growth of juvenile Japanese flounder, Paralichthys olivaceus. Aquaculture 258, 503–513.

PT E

D

Faudzi, N.M., Yong, A.S.K., Shapawi, R., Senoo, S., Biswas, A., Takii, K., 2018. Soy protein concentrate as an alternative in replacement of fish meal in the feeds of hybrid grouper,

CE

brown-marbled grouper (Epinephelus fuscoguttatus) × giant grouper (E. lanceolatus) juvenile. Aquacult. Res. 49, 431-441.

AC

Forster, I., Higgs, D.A., Dosanjh, B.S., Rowshandeli, M., Parr, J., 1999. Potential for dietary phytase to improve the nutritive value of canola protein concentrate and decrease phosphorus output in rainbow trout (Oncorhynchus mykiss) held in 11°C fresh water. Aquaculture 179, 109–125. Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture 199, 197–227.

ACCEPTED MANUSCRIPT

Furukawa, A., Tsukahara, H., 1966. On the acid digestion for the determination of chromic oxide as an index substance in the study of digestibility of fish feed. Bull. Japan Soc. Sci. Fish. 32, 502-506.

PT

Gatlin, D.M., Barrows, F.T., Brown, P., Dabrowski, K., Gaylord, T.G., Hardy, R.W., Herman, E.J., Hu, G.S., Krogdahl, Å., Nelson, R., Overturf, K., Rust, M., Sealey, W., Skonberg, D.,

SC

RI

Souza, E.J., Stone, D., Wilson, R., Wurtele, E., 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquacult. Res. 38, 551–579.

MA

Chinook salmon. J. Nutr. 62, 225-243.

NU

Halver, J.E., 1957. Nutrition of salmonid fish-III. Water-soluble vitamin requirements of

Hodge, J.E., Hofreiter, B.Y., 1962. Determination of reducing sugars and carbohydrates. In (Eds.), Methods in carbohydrate chemistry, NY, USA,

PT E

D

Whistler, R. L., Wolfrom, M. L. Academic Press, pp. 380-394.

CE

Hien, T.T.T., Be, T.T., Lee, C.M., Bengtson, D.A., 2015. Development of formulated diets for snakehead (Channa striata and Channa micropeltes): Can phytase and taurine

340.

AC

supplementation increase use of soybean meal to replace fish meal? Aquaculture 448, 334-

Jackson, L.S., Li, M.H., Robinson, E.H., 1996. Use of microbial phytase in channel catfish Ictalurus punctatus diets to improve utilization of phytate phosphorus1. J.

World

Aquacult. Soc. 27, 309–313. Kader, M.A., Koshio, S., Ishikawa, M., Yokoyama, S., Bulbul, M., 2010. Supplemental effects

ACCEPTED MANUSCRIPT

of some crude ingredients in improving nutritive values of low fishmeal diets for red sea bream, Pagrus major. Aquaculture 308, 136–144. Kader, M.A., Bulbul, M., Koshio, S., Ishikawa, M., Yokoyama, S., Nguyen, B.T., Komilus,

PT

C.F., 2012. Effect of complete replacement of fish meal by dehulled soybean meal with crude attractants supplementation in diets for red sea bream, Pagrus major. Aquaculture

SC

RI

350–353, 109–116.

Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau, B., Laroche, M., 1995. Partial

NU

or total replacement of fish meal by soybean protein on growth, protein utilization, potential

MA

estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout, Oncorhynchus mykiss. Aquaculture 133, 257-274.

PT E

D

Kissil, G.W., Lupatsch, I., Higgs, D.A., Hardy, R.W., 2000. Dietary substitution of soy and rapeseed protein concentrates for FM, and their effects on growth and nutrient utilization

CE

in gilthead seabream Sparus aurata L. Aquacult. Res. 31, 595–601. Krogdahl, Å., Penn, M., Thorsen, J., Refstie, S., Bakke, A.M., 2010. Important antinutrients in

AC

plant feedstuffs for aquaculture: an update on recent findings regarding responses in salmonids. Aquacult. Res. 41, 333–344. Kumar, V., Sinha, A.K., Makkar, H.P.S., De Boeck, G., Becker, K., 2012. Phytate and phytase in fish nutrition. J. Anim. Physiol. Anim. Nutr. 96, 335–364.

ACCEPTED MANUSCRIPT

Lanari, D., D'Agaro, E., Turri, C., 1998. Use of nonlinear regression to evaluate the effects of phytase enzyme treatment of plant protein diets for rainbow trout (Oncorhynchus mykiss). Aquaculture 161, 345–356.

PT

Lemaire, P., Drai, P., Mathieu, A., Lemarie, S., Carrière, S., Giudicelli, J., Lafaurie, M., 1991. Changes with different diets in plasma enzymes (GOT, GPT, LDH, ALP) and plasma

SC

RI

lipids (cholesterol, triglycerides) of sea-bass (Dicentrarchus labrax). Aquaculture 93, 63– 75.

NU

Liebert, F., Portz, L., 2005. Nutrient utilization of Nile tilapia Oreochromis niloticus fed plant

MA

based low phosphorus diets supplemented with graded levels of different sources of microbial phytase. Aquaculture 248, 111–119.

PT E

D

Lim, S.R., Choi, S.M., Wang, X.J., Kim, K.W., Shin, I.S., Min, T.S., Bai, S.C., 2004. Effects of dehulled soybean meal as a fish meal replacer in diets for fingerling and growing Korean

CE

rockfish, Sebastes schlegeli. Aquaculture 231, 457–468. Masumoto, T., Tamura, B., Shimeno, S., 2001. Effects of phytase on bioavailability of

AC

phosphorus in soybean meal-based diets for Japanese flounder Paralichthys olivaceus. Fish. Sci. 67, 1075–1080. Mitchell, D.B., Vogel, K., Weimann, B.J., Pasamontes, L., van Loon, A.P.G.M., 1997. The phytase subfamily of histidine acid phosphatases: Isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila. Microbiology 143, 245–252.

ACCEPTED MANUSCRIPT

Nagel, F., von Danwitz, A., Tusche, K., Kroeckel, S., van Bussel, C.G.J., Schlachter, M., Adem, H., Tressel, R.P., Schulz, C., 2012. Nutritional evaluation of rapeseed protein isolate as fish meal substitute for juvenile turbot (Psetta maxima L.) - impact on growth performance,

PT

body composition, nutrient digestibility and blood physiology. Aquaculture 356, 357–364. Papatryphon, E., Howell, R.A., Soares Jr., J.H., 1999. Growth and mineral absorption by

SC

RI

striped bass Morone saxatilis fed a plant feedstuff based diet supplemented with phytase. J. World Aquac. Soc. 30, 161–173.

NU

Papatryphon, E., Soares, J.H, 2001. The effect of phytase on apparent digestibility of four

MA

practical plant feedstuffs fed to striped bass, Morone saxatilis. Aquacult. Nutr. 7, 161–168. Peres, H., Lim, C., Klesius, P.H., 2003. Nutritional value of heat-treated soybean meal for

PT E

D

channel catfish (Ictalurus punctatus). Aquaculture 225, 67–82. Qiu, X., Davis, D.A., 2016. Effects of dietary phytase supplementation on growth performance

CE

and apparent digestibility coefficients of Pacific white shrimp Litopenaeus vannamei. Aquacult. Nutr. 23, 942-951.

AC

Refstie, S., Sahlström, S., Bråthen, E., Baeverfjord, G., Krogedal, P., 2005. Lactic acid fermentation eliminates indigestible carbohydrates and antinutritional factors in soybean meal for Atlantic salmon (Salmo salar). Aquaculture 246, 331–345. Rodehutscord, M., Pfeffer, E., 1995. Effects of supplemental microbial phytase on phosphorus digestibility and utilization in rainbow trout (Oncorhynchus mykiss). Water Sci. Technol. 31, 143–147.

ACCEPTED MANUSCRIPT

Sajjadi, M., Carter, C.G., 2004. Dietary phytase supplementation and the utilization of phosphorus by Atlantic salmon (Salmo salar L.) fed a canola-meal-based diet. Aquaculture 240, 417-431.

PT

Salze, G., Mclean, E., Battle, P.R., Schwarz, M.H., Craig, S.R., 2010. Use of soy protein concentrate and novel ingredients in the total elimination of fish meal and fish oil in diets

SC

RI

for juvenile cobia, Rachycentron canadum. Aquaculture 298, 294–299.

Silva-Carrillo, Y., Hernández, C., Hardy, R.W., González-Rodríguez, B., Castillo-

NU

Vargasmachuca, S., 2012. The effect of substituting fish meal with soybean meal on

MA

growth, feed efficiency, body composition and blood chemistry in juvenile spotted rose snapper Lutjanus guttatus (Steindachner, 1869). Aquaculture 364-365, 180.185.

PT E

D

Storebakken, T., Shearer, K.D., Roem, A.J., 1998. Availability of protein, phosphorus and other elements in fish meal, soy-protein concentrate and phytase-treated soy-protein-

CE

concentrate-based diets to Atlantic salmon, Salmo salar. Aquaculture 161, 365–379. Sugiura, S.H., Raboy, V., Young, K.A., Dong, F.M., Hardy, R.W., 1999. Availability of

AC

phosphorus and trace elements in low-phytate varieties of barley and corn for rainbow trout (Oncorhynchus mykiss). Aquaculture, 170, 285–296. Sugiura, S.H., Gabaudan, J., Dong, F.M., Hardy, R.W., 2001. Dietary microbial phytase supplementation and the utilization of phosphorus, trace minerals and protein by rainbow trout [Oncorhynchus mykiss (Walbaum)] fed soybean meal-based diets. Aquac. Res. 32, 583–592.

ACCEPTED MANUSCRIPT

Takagi, S., Hosokawa, H., Shimeno, S., Maita, M., Ukawa, M., Ueno, S., 1999. Utilization of soy protein concentrate in a diet for red sea bream, Pagrus major. Suisanzoshoku 47, 77– 87.

PT

Takagi, S., Shimeno, S., Hosokawa, H., Ukawa, M., 2000. Replacement of fish meal by combined inclusion of alternative protein sources in a diet for yearling red sea bream,

SC

RI

Pagrus major. Suisanzoshoku 48, 545–552.

Takagi, S., Shimeno, S., Hosokawa, H., Ukawa, M., 2001. Effect of lysine and methionine

NU

supplementation to a soy protein concentrate diet for red sea bream Pagrus major. Fish.

MA

Sci. 67, 1088–1096.

Tantikitti, C., Sangpong, W., Chiavareesajja, S., 2005. Effects of defatted soybean protein

PT E

D

levels on growth performance and nitrogen and phosphorus excretion in Asian seabass (Lates calcarifer). Aquaculture 248, 41–50.

CE

Uyan, O., Koshio, S., Teshima, S., Ishikawa, M., Michael, F.R., Ren, T., Laining, A., 2007. Effects of tuna muscle powder in diet on the growth and phosphorus loading of juvenile

AC

red sea bream, Pagrus major. Aquacult. Sci. 55, 29–40. Van der Ingh, T.S.G.A.M., Krogdahl, A., Olli, J.J., Hendriks, H.G.C.J.M., Koninkx, J.F.J.G., 1991. Effects of soybean-containing diets on the proximal and distal intestine in Atlantic salmon (Salmo salar): a morphological study. Aquaculture 94, 297–305.

ACCEPTED MANUSCRIPT

Vielma, J., Mäkinen, T., Ekholm, P., Koskela, J., 2000. Influence of dietary soy and phytase levels on performance and body composition of large rainbow trout (Oncorhynchus mykiss) and algal availability of phosphorus load. Aquaculture 183, 349–362.

PT

Vielma, J., Ruohonen, K., Peisker, M., 2002. Dephytinization of two soy proteins increases phosphorus and protein utilization by rainbow trout, Oncorhynchus mykiss. Aquaculture

SC

RI

204, 145-156.

Wang, L., Zhou, H., He, R., Xu, W., Mai, K., He, G., 2016. Effects of soybean meal

NU

fermentation by Lactobacillus plantarum P8 on growth, immune responses, and intestinal

MA

morphology in juvenile turbot (Scophthalmus maximus L.). Aquaculture 464, 87-94. Wilson, R.P., Poe, W.E., 1985. Effects of feeding soybean meal with varying trypsin inhibitor

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D

activities on fingerling channel catfish. Aquaculture 46, 19–25. Yamamoto, T., Goto, T., Kine, Y., Endo, Y., Kitaoka, Y., Sugita, T., Furuita, H., Iwashita, Y.,

CE

Suzuki, N., 2008. Effect of an alcohol extract from a defatted soybean meal supplemented with a casein-based semi-purified diet on the biliary bile status and intestinal conditions

AC

in rainbow trout Oncorhynchus mykiss (Walbaum). Aquacult. Res. 39, 986–994. Yamamoto, T., Iwashita, Y., Matsunari, H., Sugita, T., Furuita, H., Akimoto, A., Okamatsu, K., Suzuki, N., 2010. Influence of fermentation conditions for soybean meal in a non-fish meal diet on the growth performance and physiological condition of rainbow trout Oncorhynchus mykiss. Aquaculture 309, 173–180.

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Yoo, G.Y., Wang, X., Choi, S., Han, K., Kang, J.C., Bai, S.C., 2005. Dietary microbial phytase increased the phosphorus digestibility in juvenile Korean rockfish Sebastes schlegeli fed diets containing soybean meal. Aquaculture 243, 315–322.

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Zhang, Y., Wu, Y.B., Jiang, D.L., Qin, J.G., Wang, Y., 2014. Gamma-irradiated soybean meal replaced more fish meal in the diets of Japanese seabass (Lateolabrax japonicus). Anim.

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RI

Feed Sci. Technol. 197, 155-163.

Zhu, Y., Qiu, X., Ding, Q.L., Duan, M.M., Wang, C.F., 2014. Combined effects of dietary

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phytase and organic acid on growth and phosphorus utilization of juvenile yellow catfish

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Pelteobagrus fulvidraco. Aquaculture 430, 1–8.

ACCEPTED MANUSCRIPT Table 1 Dietary formula and proximate composition (%) for Trial 1. C

SPC60

SPC70

SPC80

SPC100

67.0

26.8

20.1

13.5

SPCb

35.0

40.0

45.0

55.0

Corn gluten

9.0

10.0

11.0

13.0

9.5

10.0

11.0

3.0

3.0

3.0

3.0

3.0

3.0

7.0

9.0

α‐Starch

10.0

3.0

Vitamin mixturec

3.0

3.0

Mineral mixturec

5.0

7.0

8.0

9.0

11.0

2.0

2.0

2.0

2.0

4.0

2.9

1.8

0.0

0.7

1.0

1.2

1.5

0.5

0.5

0.5

0.5

50.1

50.7

50.1

49.9

47.8

15.1

15.9

15.7

15.6

15.7

13.7

12.5

12.3

12.3

11.2

11.5

14.3

15.3

16.0

16.6

19.4

20.1

20.0

20.2

19.9

Soybean lecithin 7.5

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Cellulose Taurine Chromic oxide (Cr2O3)

0.5

RI

Fish oil

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Fish meala

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Feed formula (%)

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Proximate analysis (% of dry matter basis)

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Crude protein Crude lipid

Crude sugar

a

Chubu Feed Co. Ltd., Nagoya, Japan (ingredient: sardine; protein 67%).

b c

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Energy(kJ/g)

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Crude ash

Soy protein concentrate (SPC, protein 63%), Profit 1000, Fuji Oil, Osaka, Japan.

Halver (1957).

ACCEPTED MANUSCRIPT Table 2 Feed formula and proximate composition for Trial 2. Ingredients

C

SPC80

P1

P2

P3

P4

67.0

13.5

13.5

13.5

13.5

13.5

SPCb

-

45.0

45.0

45.0

45.0

45.0

Corn gluten

-

11.0

11.0

11.0

11.0

11.0

Fish oil

7.0

10.0

10.0

10.0

10.0

10.0

α-Starch

10.0

3.0

3.0

3.0

3.0

3.0

Vitamin mixuturec

3.0

3.0

3.0

3.0

3.0

3.0

Mineral mixuturec

5.0

9.0

9.0

9.0

9.0

9.0

2.0

2.0

2.0

2.0

2.0

1.8

1.8

1.8

1.8

1.8

Soybean lecithin

7.5

MA

Cellulose Taurine

RI

SC

NU

Fish meala

PT

Feed formula (%)

-

1.2

1.2

1.2

1.2

1.2

0.5

0.5

0.5

0.5

0.5

0.5

-

-

0.02

0.04

0.06

0.08

51.1

49.8

50.1

50.1

50.0

50.3

15.9

15.3

15.3

15.1

15.7

15.00

12.3

12.0

11.9

11.8

11.0

11.9

11.4

15.9

16.0

16.0

15.7

15.9

Phosphorus (g/kg diet)

20.6

13.5

13.5

12.9

13.3

13.8

Energy (kJ/g diet)

20.4

21.2

21.2

21.1

21.2

20.9

Chromic oxide (Cr2O3)

D

Phytased

PT E

Proximate composition (%, dry matter basis) Crude protein

Crude ash

AC

Crude sugar

CE

Crude lipid

a

Chubu Feed Co. Ltd., Nagoya, Japan (ingredient: sardine; protein 67%).

b c

Soy protein concentrate (SPC, protein 63%), Profit 1000, Fuji Oil, Osaka, Japan.

Halver (1957).

d

BASF, Tokyo, Japan (5000 FTU/g)

ACCEPTED MANUSCRIPT Table 3 Indispensable (IAA) and non-indispensable (NIAA) amino acid composition (g/100 g dry basis) of ingredients and experimental diets fed to red sea bream in Trial 1. Ingredients Fish

SPC

Diets Corn

Amino acids meal

C

SP60

SP70

SP80

SP100

2.78 (13.8)

2.69 (14.5)

1.29 (6.4)

1.23 (6.2)

1.09 (5.9)

gluten

4.05

4.40

2.10

2.75 (12.5)*

2.81 (13.6)

Histidine

2.44

1.65

1.40

1.63 (7.5)

1.36 (6.6)

Isoleucine

2.97

2.67

2.64

1.99 (9.1)

1.97 (9.5)

PT

2.76 (14.0)

1.93 (9.6)

1.89 (9.6)

1.81 (9.8)

Leucine

5.21

4.53

10.20

3.49 (16.0)

3.90 (18.8)

3.88 (19.2)

3.86 (19.6)

3.82 (20.6)

Lysine

5.68

3.75

1.16

3.81 (17.5)

2.94 (14.2)

2.76 (13.7)

2.58 (13.1)

2.21 (11.9)

Methionine

1.82

0.78

1.59

1.22 (5.6)

0.90 (4.3)

0.84 (4.1)

0.77 (3.9)

0.64 (3.4)

Phenylalanine

2.85

3.03

4.12

1.91 (8.8)

2.20 (10.6)

2.20 (10.9)

2.20 (11.2)

2.20 (11.9)

Threonine

3.05

2.37

2.25

2.04 (9.4)

1.85 (8.9)

1.79 (8.8)

1.73 (8.8)

1.60 (8.6)

Tryptophan

0.87

0.85

0.40

0.58 (2.7)

0.57 (2.7)

0.55 (2.7)

0.54 (2.8)

0.52 (2.8)

Valine

3.57

2.88

2.99

2.39 (11.0)

2.23 (10.8)

2.17 (10.7)

2.11 (10.7)

1.97 (10.6)

32.51

26.91

28.85

21.78 (100)

20.73 (100)

20.18 (100)

19.67 (100)

18.55 (100)

88.2

88.2

84.0

83.7

NU

D

IAA index

MA

ƩIAA

PT E

NIAA

RI

Arginine

SC

IAA

4.35

2.55

5.71

2.91 (13.5)

2.57 (10.9)

2.47 (10.5)

2.36 (10.2)

2.14 (9.3)

Aspartic acid

6.42

6.70

4.10

4.30 (20.0)

4.43 (18.9)

4.38 (18.7)

4.33 (18.6)

4.22 (18.4)

Cystine

0.71

0.92

1.25

0.48 (2.2)

0.62 (2.6)

0.64 (2.7)

0.65 (2.8)

0.67 (2.9)

Glutamic acid

8.87

11.30

14.10

5.94 (27.6)

7.60 (32.3)

7.71 (33.0)

7.83 (33.7)

8.05 (35.1)

Glycine

4.03

2.52

1.87

2.70 (12.5)

2.13 (9.1)

2.01 (8.6)

1.88 (8.1)

1.63 (7.1)

2.76

3.10

6.10

1.85 (8.6)

2.37 (10.1)

2.40 (10.3)

2.44 (10.5)

2.50 (10.9)

2.71

3.10

3.37

1.82 (8.4)

2.11 (9.0)

2.12 (9.1)

2.13 (9.2)

2.14 (9.3)

2.32

2.14

3.32

1.55 (7.2)

1.67 (7.1)

1.65 (7.1)

1.64 (7.1)

1.61 (7.0)

32.17

32.33

39.82

21.55 (100)

23.50 (100)

23.38 (100)

23.27 (100)

22.96 (100)

Serine Tyrosine ƩNIAA

AC

Proline

CE

Alanine

*Data in parenthesis indicate contribution of each fatty acid to total IAA or NIAA in the experimental diets.

ACCEPTED MANUSCRIPT Table 4 Growth performance of red sea bream fed with different diets for 10 weeks in Trial 1. SPC60

SPC70

SPC80

SPC100

Initial body weight (ｇ)

23.1±0.2

23.2±0.2

22.9±0.3

22.8±0.2

23.2±0.2

Final body weight (g)

87.6±2.9ª

87.1±3.3ª

80.0±4.2ª

69.3±4.3ｂ

50.6±7.6c

Survival rate (%）

97.8±1.9a

97.8±1.9a

95.6±3.9a

98.9±1.9a

81.1±1.9b

SGR (%/day)

1.9±0.1a

1.9±0.1a

1.8±0.1a

1.6±0.1ab

1.1±0.1b

Total feed intake (g)

2550±38a

2726±129a

2494±145a

2087±208a

1262±12b

FE (%)

74.0±0.9a

69.4±2.2ab

66.1±4.4ab

65.9±1.2b

51.3±4.8c

CF

3.3±0.2a

3.3±0.1a

3.1±0.1ab

3.0±0.2ab

2.9±0.2b

RI

SC

NU

Each value is a mean ± SD (n = 3).

PT

C

AC

CE

PT E

D

MA

Values in a row with different superscripts are significantly different (P < .05, Tukey's test). SGR, specific growth rate; DFR, daily feeding rate; FE, feed efficiency; CF, condition factor.

ACCEPTED MANUSCRIPT Table 5

Proximate composition of whole body, apparent digestibility and retention efficiency in fish under different treatments in Trial 1. Final Initial

C

SPC60

SPC70

SPC80

SPC100

PT

Proximate composition (%) 70.7±0.2

65.6±0.5a

65.6±0.8a

66.3±1.0a

65.9±1.6a

70.4±1.8b

Crude protein

17.9±0.5

16.9±0.2a

17.4±0.4a

17.0±0.4a

17.1±0.4a

15.6±0.4b

Crude lipid

6.2±0.1

12.6±0.4a

12.6±0.9a

11.5±1.0ab

10.8±0.5b

8.7±0.6c

Crude ash

5.0±0.2

4.8±0.2

4.3±0.5

4.6±0.4

5.1±0.2

5.1±0.5

94.6±1.9

91.3±1.7

SC

NU

95.1±2.0

Protein

28.6±0.2a

28.3±2.1a

27.0±2.9a

25.8±3.3a

17.2±3.7b

Lipid

80.9±4.1a

80.7±5.3a

76.6±3.9a

70.0±5.7a

46.5±6.7b

Energy

D

Apparent digestibility (%)

RI

Moisture

37.5±1.7a

35.8±2.9a

30.4±2.7ab

29.4±3.1b

20.4±3.0c

94.4±3.1

Protein

PT E

MA

Retention efficiency (%)

95.8±2.2

Each value is a mean ± SD (n = 3).

AC

CE

Values in a row with different superscripts are significantly different (P < .05, Tukey's test).

ACCEPTED MANUSCRIPT Table 6 Relative organ weight and plasma constituents in fish fed with different diets in Trial 1. Final Initial

C

SPC60

SPC70

SPC80

SPC100

8.8

8.9±0.4a

8.7±0.4ab

8.3±0.5ab

8.5±0.8ab

7.7±0.8b

HSI

1.8

1.9±0.1a

1.7±0.1ab

1.8±0.5a

1.7±0.3ab

1.1±0.2b

SSI

0.6

0.7±0.1

0.7±0.1

0.8±0.1

0.8±0.1

0.9±0.1

ISI

1.1

1.1±0.1

1.3±0.1

1.3±0.2

1.2±0.2

1.5±0.3

GOT (U/l)

30.0±5.3

36.4±5.3

33.3±4.6

33.3±5.1

GPT (U/l)

13.7±2.6

19.0±3.3

14.2±2.9

10.5±3.3

18.3±5.1

TP (g/dl)

4.8±0.9

4.3±0.7

3.7±1.1

3.6±0.9

3.7±0.5

174.7±17.1

132.3±12.8

190.0±18.1

187.3±17.2

RI

39.1±6.8

MA

NU

Plasma constituents

PT

VSI

SC

Relative organ weight (%)

155.3±16.3

TCHO (mg/dl)

380.7±29.3a

288.7±31.1ab

239.7±27.3b

259.7±23.9b

215.3±25.5b

66.7±4.9

78.2±7.1

65.7±4.3

60.3±3.9

61.7±4.4

PT E

GLU (mg/dl)

D

TG (mg/dl)

Each value is a mean ± SD (n = 3).

Values in a row with different superscripts are significantly different (P < .05, Tukey's test).

CE

VSI, viscerosomatic index; HIS, hepatosomatic index; SSI, stomatosomatic index; ISI,

AC

intestinosomatic index; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; TP, total protein; TG, triglyceride; TCHO, total cholesterol; GLU, glucose.

ACCEPTED MANUSCRIPT

SPC80

P1

P2

P3

P4

Initial body weight (g)

21.1±0.5

20.9±0.4

21.3±0.6

21.0±0.3

21.3±0.3

21.9±0.6

Final body weight (g)

94.8±3.3a

70.8±4.2c

77.6±3.5b

77.9±3.2b

76.0±8.1bc

76.4±7.4bc

Survival rate (%）

93.3±7.6

98.3±2.9

91.7±2.9

91.7±7.6

90.0±5.0

90.0±13.2

SGR (%/day)

2.2±0.1a

1.7±0.2b

1.8±0.2b

1.8±0.4b

1.8±0.3b

1.8±0.3b

DFR (%)

2.1±0.1b

2.6±0.1a

2.6±0.1a

2.6±0.2a

2.6±0.2a

2.5±0.2a

FE (%)

83.5±1.1a

63.7±1.9c

70.8±3.2b

70.9±2.6b

69.1±10.4bc

70.2±6.7bc

3.3±0.4

3.2±0.5

3.3±0.4

3.2±0.2

3.2±0.4

3.2±0.3

SC

CF

PT

C

RI

Table 7 Growth performance of red sea bream fed with different diets for 10 weeks in Trial 2.

NU

Each value is a mean ± SD (n = 3).

Values in a row with different superscripts are significantly different (P < .05, Tukey's test).

AC

CE

PT E

D

MA

SGR, specific growth rate; DFR, daily feeding rate; FE, feed efficiency; CF, condition factor.

ACCEPTED MANUSCRIPT Table 8 Proximate composition of whole body, apparent digestibility and retention efficiency in fish under different treatments in Trial 2. Final Initial C

SPC80

P1

P2

P3

P4

PT

Proximate composition (%) 69.5±0.3

60.7±0.5

62.7±1.8

64.1±1.4

60.9±1.8

62.7±2.8

64.1±2.8

Crude protein

17.3±0.4

17.6±0.3

16.4±0.4

16.5±0.3

17.0±0.7

17.0±0.4

16.7±0.7

Crude lipid

7.6±0.2

14.8±0.2a

12.0±0.9b

13.2±0.8ab

13.9±0.7ab

13.5±0.9ab

13.1±0.8ab

Crude ash

4.9±0.2

5.7±0.1

6.7±0.3

Phosphorus (g/kg)

4.8±0.2

4.8±0.3

3.8±0.2

Protein

94.3±2.1

Energy

89.8±3.1

Phosphorus

66.9±1.7b

SC

5.9±0.5

5.6±0.4

5.9±0.5

4.6±0.4

4.6±0.5

4.7±0.3

4.4±0.5

91.2±2.1

93.3±1.9

94.8±2.2

93.9±1.5

94.1±3.1

87.5±2.9

88.2±3.2

89.7±3.5

89.2±2.9

89.1±2.7

68.5±1.5b

73.9±1.1a

75.3±1.3a

74.4±1.8a

77.1±2.1a

NU

5.8±0.4

Energy

AC

Phosphorus

D

CE

Lipid

PT E

Protein

MA

Apparent digestibility (%)

Retention efficiency (%)

RI

Moisture

31.3±0.1a

24.4±1.3c

25.7±1.0c

27.8±1.3b

26.4±1.7bc

26.3±1.1bc

88.9±3.2a

64.4±4.7c

68.4±3.1bc

70.2±3.7b

69.1±2.6bc

70.2±2.9b

41.2±1.0a

28.9±2.1b

29.9±1.7b

33.1±2.5b

30.7±2.2b

30.1±1.9b

19.6±1.8a

12.6±1.7b

18.8±1.8a

19.4±2.9a

17.7±1.7a

16.7±2.5a

Each value is a mean ± SD (n = 3). Values in a row with different superscripts are significantly different (P < .05, Tukey's test).

ACCEPTED MANUSCRIPT Table 9 Relative organ weight and plasma constituents in fish fed with different diets in Trial 2.

C

SPC80

P1

P2

P3

P4

8.8±0.5

8.0±0.4

7.7±0.6

8.1±0.4

8.3±0.6

8.1±0.4

HSI

1.5±0.1

1.6±0.2

1.4±0.1

1.4±0.2

1.5±0.2

1.7±0.3

SSI

0.7±0.1

0.6±0.1

0.7±0.1

0.6±0.1

0.5±0.2

0.6±0.1

ISI

0.7±0.1

0.7±0.1

0.8±0.2

0.7±0.1

0.6±0.1

0.7±0.1

GOT (U/l)

39.3±7.9

61.3±12.3

53.6±13.3

64.7±15.0

63.1±14.3

65.4±13.7

GPT (U/l)

10.4±3.1

17.9±4.1

11.0±2.9

14.3±4.0

11.8±2.9

11.2±2.8

TP (g/dl)

4.2±0.5

4.0±0.4

4.3±1.0

5.2±1.3

5.1±1.1

4.3±0.6

TG (mg/dl)

176.1±15.4

137.6±12.3

169.6±13.3

110.1±10.1

141.1±9.9

142.3±11.9

TCHO (mg/dl)

263.2±22.6a

164.4±12.9b

196.0±17.7ab

207.0±20.3ab

190.5±25.4ab

188.6±24.3ab

GLU (mg/dl)

60.8±5.3

68.2±6.3

75.4±4.7

67.3±4.1

63.7±4.0

64.5±3.9

RI

D

MA

SC

Plasma constituents

PT

VSI

NU

Relative organ weight (%)

PT E

Each value is a mean ± SD (n = 3).

Values in a row with different superscripts are significantly different (P < .05, Tukey's test). VSI, viscerosomatic index; HSI, hepatosomatic index; SSI, stomatosomatic index; ISI,

CE

intestinosomatic index; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic

AC

transaminase; TP, total protein; TG, triglyceride; TCHO, total cholesterol; GLU, glucose.

ACCEPTED MANUSCRIPT

Optimum fish meal replacement by soy protein concentrate from soymilk and phytase supplementation in diets of red sea bream, Pagrus major

SC

RI

PT

Amal Biswas*, Hideo Araki, Tetsuo Sakata, Toshihiro Nakamori, and Kenji Takii

Highlights

NU

●This study aimed to determine if soy protein concentrate (SPC) derived from soymilk rather

MA

than soybean meal could replace more fish meal (FM) without supplementation of AAs or palatability enhancers in the diet of red sea bream.

PT E

D

● SPC derived from soymilk used in this study contentedly replaced 70% of FM, which could be beneficial to the aquaculture industry of red sea bream.

CE

●Although phytase supplementation in the diet ensures environmental benefit for industry use in aquaculture production through reduction in P-discharge, growth performance did not

AC

improve to the level of control group when SPC from soymilk replaced FM. ●The results open a new window to establish a diet for red sea bream, which will help to achieve sustainability of the aquaculture industry of this species.