# Evaluation of Six Selected Commercial Fermented Soybean Meal by Feeding Juvenile Turbot (Scophthalmus maximus L.)

Authors: Jichang Zheng; Zhijie Dan; Dali Jiang; Zhuoyi Tang; Si Zhu; Qingfei Li; Xueshan Li; Kangsen Mai; Qinghui Ai
Journal: Aquaculture Nutrition
 (2022)

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## Abstract

Various kinds of fermented soybean meal are now commercially available, whereas the quality of these products is uneven due to different processing technologies, fermentation strains, and raw soybean meal, which would lead to different effects on bioavailability of nutrients in aquaculture feeds. Thus, a 10-week feeding trial was conducted to evaluate the effects of six different commercially available fermented soybean meal (FSBM) on growth performance of juvenile turbot (Scophthalmus maximus L.) and to analyze the correlation between growth parameters and FSBM components. Seven isonitrogenous and isolipidic diets were formulated: fish-meal-based diet (the control group, FM) and FM with 450 g/kg of fish meal substituted by six commercially available FSBM (the replacement groups, FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6). Results showed that specific growth rate (SGR) and weight gain rate (WGR) were significantly higher in groups FMSB3 and FMSB4 than in other replacement groups, but significantly lower in the control group. Besides, no significant differences were observed in feed efficiency ratio (FER) and protein efficiency ratio (PER) among groups FSBM3, FSBM4, and FM. Correlation analysis revealed that SGR, PER, and protein retention (PR) of turbot were all positively correlated with the water-soluble protein and trichloroacetic acid- (TCA-) soluble protein content of FSBM, but negatively correlated with the content of trypsin inhibitor. In summary, the commercial FSBM3 and FSBM4 could yield higher growth performance of juvenile turbot than the rest selected FSBM when replacing 450 g/kg of fish meal in diets, and the content of water-soluble protein, TCA-soluble protein, and trypsin inhibitor could serve as relatively accurate indicators in the quality evaluation of FSBM.


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## Body

## 1. Introduction

Over the past few decades, increasing demand and price of fish meal have limited the expansion of aquaculture [1, 2]. To address this concern, considerable studies have been performed to evaluate the effects of replacing fish meal by other protein sources, such as poultry by-product meal [3], meat and bone meal [4], corn gluten meal [5], soybean meal [6], and cottonseed meal [7]. Among these alternative protein sources, soybean meal is a good candidate for its reasonable price, steady supply, and relatively well-balanced amino acid profile [8]. Nevertheless, the antinutritional factors (ANFs) in soybean meal cannot be ignored, since their negative effects on digestion, absorption, and utilization of nutrients by fish would impede growth performance ultimately [9]. Fermentation treatment is regarded as an effective method [10, 11] since it can reduce ANF content and improve nutrient digestibility to a great extent [12, 13]. Li et al. [14] recorded that fermentation treatment could reduce the levels of glycinin, β-conglycinin, and trypsin inhibitors by 24.2%, 22.1%, and 44.9%, respectively. Besides, fermentation treatment not only breaks up high-molecular-weight protein into polypeptide and small peptide but also produces some unknown bioactive substances that are potentially good for growth and health [15]. Nowadays, numerous kinds of fermented soybean meal (FSBM) products are commercially available on the market. However, the quality of these products is uneven due to different fermentation conditions, fermentation strains, and raw soybean meal [10], which would affect growth performance of aquatic animals. To evaluate the nutritional value of FSBM, a series of trials have been conducted to investigate the optimal levels of fish meal replaced by FSBM in different fish, such as in turbot, Scophthalmus maximus L. (450 g/kg) [16, 17], Japanese flounder, Paralichthys olivaceus (360 g/kg) [18], black sea bream, Mylio macrocephalus (200, 400 g/kg) [11, 19], rainbow trout, Oncorhynchus mykiss (600 g/kg) [20], and Japanese seabass, Lateolabrax japonicus (400 g/kg) [21].Turbot (Scophthalmus maximus L.) is widely farmed around the world, and the cultivation of which has developed into dominant mariculture industries in northern China [22]. However, turbot has a high dietary protein requirement ranging from 500 to 650 g/kg, and most of it comes from fish meal [23, 24]. Thus, it is of great economic value to choose turbot as experimental subject. Previous experiments on turbot mainly focused on the optimal level of replacing fish meal with soybean meal fermented by a single strain, such as Lactobacillus plantarum P8 [17], Aspergillus awamori [16], and Enterococcus faecium [14], whereas the information on the parallel comparison of different commercial FSBM is very scarce. Hence, this study was performed to evaluate the effects of six major commercial FSBM on the growth performance of juvenile turbot and to analyze the correlation between growth parameters and FSBM components, which would provide reference for FSBM producers to improve product quality and help aquafeed companies to select high-quality FSBM.


## 2. Materials and Methods

### 2.1. Experimental Diets

Seven isonitrogenous and isolipidic diets were formulated to contain approximately 520 g/kg crude protein and 110 g/kg crude lipid. These diets were divided into control group FM and six replacement groups FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6 (replacing 450 g/kg of fish meal with six different commercially available FSBM, which were collected from different firms in China through Qingdao Bio-ways Ingredients Bio-technology Co., Ltd.). Each diet containing FSBM was supplemented with coated L-methionine, L-isoleucine, and L-lysine to meet the requirements for juvenile turbot (Tables1 and 2). Yttrium oxide (Y2O3) was also added into each diet at the level of 1 g/kg as the indicator for dry matter and crude protein digestibility analysis. Ingredients were smashed into powder which could pass through 250 μm aperture mesh. All ingredients were thoroughly mixed with fish oil, and water was added in to produce moist dough. The moist dough was then pelleted by an experimental granulator (EL-220, Haiyang City Huatong Machinery Co., Ltd.) with 2 mm die and dried in a ventilated oven (CT-C-1, Jiang Yin Zhou Yuan Pharmaceutical Equipment Co., Ltd.) at 45°C for 10 h. All the pellets were stored at -20°C for use.Table 1
Formulation and proximate composition of experimental diets (g/kg dry matter)1.
IngredientsDiets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Brown fish meal1500.0275.0275.0275.0275.0275.0275.0Fermented soybean meal0.0300.4303.3293.2294.8293.0297.9Blood meal10.045.045.045.045.045.045.0Wheat gluten meal1134.092.090.090.090.090.092.0Wheat meal1244.5146.2142.4153.7150.9153.9146.6Fish oil45.062.062.063.064.062.064.0Soy lecithin20.020.020.020.020.020.020.0Vitamin premix315.015.015.015.015.015.015.0Vitamin C35.05.05.05.05.05.05.0Mineral premix415.015.015.015.015.015.015.0L-Methionine0.01.22.42.72.53.41.8L-Isoleucine0.01.11.10.70.90.80.8L-Lysine0.00.62.30.20.40.40.4Taurine5.05.05.05.05.05.05.0Choline chloride3.03.03.03.03.03.03.0Mildew preventive51.01.01.01.01.01.01.0Ethoxyquinoline0.50.50.50.50.50.50.5Ca(H2PO4)25.05.05.05.05.05.05.0Yttrium oxide1.01.01.01.01.01.01.0Sodium alginate1.01.01.01.01.01.01.0Attractants65.05.05.05.05.05.05.0Proximate compositionCrude protein525.5529.4522.2523.2525.1524.5529.0Crude lipid110.4109.7109.3112.1108.9109.6113.0Ash113.995.299.096.897.598.596.21Brown fish meal (g/kg dry matter): crude protein 724.7, crude lipid 98.4; blood meal (g/kg dry matter): crude protein 986.4, crude lipid 12.2; wheat gluten meal (g/kg dry matter): crude protein 791.5, crude lipid 19.3; wheat meal (g/kg dry matter): crude protein 139.3, crude lipid 24.7. These ingredients were provided by Great Seven Bio-tech (Qingdao, China). The commercial fermented soybean meal was collected by Qingdao Bio-ways Ingredients Bio-technology Co., Ltd. (Qingdao, China). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6. 3Vitamin premix (mg/kg diet): retinol acetate, 32; cholecalciferol, 5; alpha-tocopherol, 240; thiamin, 25; riboflavin, 45; pyridoxine HCl, 20; vitamin B12, 10; pantothenic acid, 60; folic acid, 20; niacin 200; biotin, 60; inositol, 800; microcrystalline cellulose, 13,473. Vitamin C was supplied in the form of vitamin C polyphosphate. 4Mineral premix (mg/kg diet): MgSO4·7H2O, 1200; CuSO4·5H2O, 10; FeSO4·H2O, 80; ZnSO4·H2O, 50; MnSO4·H2O, 45; CoCl2·6H2O, 50; Na2SeO3, 20; H2CaIO4, 60; zeolite powder, 13,485. 5Mildew preventive: calciumpropionate:boleticacid=1:1. 6Attractants: glycine betaine : DMPT : glycine : alanine : inosine-5-diphosphate trisodium salt=4:2:2:1:1.Table 2
Amino acid composition of experimental diets (g/kg dry matter)1.
Amino acidsDiets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Essential amino acidThreonine19.119.117.919.320.218.918.5Valine24.026.026.426.628.925.826.4Methionine12.211.713.013.513.714.111.8Isoleucine22.121.722.022.324.122.021.5Leucine36.639.339.441.043.940.139.1Phenylalanine22.725.225.926.728.225.224.1Lysine28.127.228.129.130.828.027.2Histidine14.114.815.015.516.715.014.7Arginine25.225.726.127.629.127.026.8Nonessential amino acidProline26.024.123.724.225.624.524.0Aspartic acid36.641.841.744.547.643.943.3Serine20.821.421.022.823.722.621.4Glutamic acid104.196.293.598.8106.097.594.9Glycine29.227.027.327.429.427.526.3Alanine30.130.429.330.232.429.929.0Cysteine5.35.25.45.76.35.85.4Tyrosine15.615.616.816.817.815.915.11Data are means of triplicate. No tryptophan was detected because of acid hydrolysis. 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6.


### 2.2. Experimental Fish and Procedure

Juvenile turbot were purchased from a commercial hatchery in Weihai, China. The feeding trial was conducted in LaiZhou MingBo Aquatic Co., Ltd. (Yantai, China). Turbot were fed with commercial diets for 14 days to acclimate to the experimental conditions. At the start of the feeding trial, all the turbot were fasted for 24 h and some of the fish were randomly weighed to estimate the average weight of the batch. Afterwards, fish of similar weight (6.99±0.02g) were selected and randomly assigned into 21 experimental fibreglass tanks (600 L capacity). Each diet was randomly assigned to triplicate tanks. Fish were fed twice daily (08:00 and 18:00) to apparent satiation for 10 weeks. After feeding, feces waste was cleaned up and seawater was also renewed. During the feeding trial, the water temperature ranged from 18 to 20°C, salinity ranged from 30 to 32, and dissolved oxygen maintained at approximately 6.0 mg/L. Meanwhile, the photoperiod was maintained on 12 h light : 12 h dark.


### 2.3. Sample Collection

At the start of formal growth experiment, twenty fish were randomly sampled from the remaining turbot for initial carcass protein analysis. From the seventh week of the growth experiment, feces samples were collected by siphoning after 8 h of feeding. All the collected feces samples were stored at -20°C for analysis. At the termination of the feeding trial, all the fish were fasted for 24 h and anesthetized with eugenol (1 : 12,000) to reduce the stress reaction before sampling. Then, the total number and weight of fish in each tank were first recorded. Six fish were randomly sampled from each tank to measure individual body weight, body length, visceral weight, and liver weight. Intact intestines were sampled from four fish per tank and frozen in liquid nitrogen immediately and then stored at -80°C for digestive enzyme analysis. Moreover, the middle intestine was sampled from three fish each tank for histology analysis. The sampled middle intestine (1 cm in length) were placed in 4% paraformaldehyde solution for fixation and then transferred to 75% ethanol after 24 h.


### 2.4. Biochemical Analysis

Proximate composition of ingredients, diets, and feces was analyzed according to published standards [25]. Moisture was determined by drying samples to a constant weight at 105°C for 24 h. Ash was measured gravimetrically by placing the samples in muffle furnace at 550°C for 16 h after ignition. Crude lipid was analyzed by petroleum ether extraction using Soxhlet (Buchi 36680, Switzerland). Crude protein was analyzed by the Kjeldahl method (Kjeltec™ 8400, FOSS, Sweden). Amino acids of ingredients and diets were analyzed by an amino acid analyzer (L-8900, Hitachi) after acid hydrolysis (6 N HCl for 24 h at 110°C). The content of water-soluble protein, trichloroacetic acid- (TCA-) soluble protein, and alkali- (KOH-) soluble protein was determined according to the methods described by Sarin [26] and Araba and Dale [27] with some modifications. Among them, the water-soluble protein in soybean meal was determined after three times of ultrapure water extraction in a shaker. For the determination of TCA-soluble protein, soybean meal was extracted by 15% trichloroacetic acid for 5 min, and supernatant was centrifuged (4000 r/min, 5 min) for protein determination. As to the analysis of alkali-soluble protein, soybean meal was extracted by 0.2% potassium hydroxide for 20 min in a magnetic stirrer; the supernatant used for protein determination was then obtained after centrifugation (2700 r/min, 10 min). The total calcium and phosphorus contents of these six FSBM were measured by an inductively coupled plasma-atomic emission spectrophotometer (ICP-OES, Thermo Fisher 7200).


### 2.5. Antinutritional Factor Analysis

The trypsin inhibitor, glycinin, andβ-conglycinin contents of FSBM were all analyzed by a microplate reader (SpectraMax i3, Molecular Devices) using competitive enzyme-linked immunosorbent assay (ELISA) kits (Beijing Longkefangzhou Bio-Engineering Technology Co., Ltd., China); all procedures performed in the analysis were in strict accordance with the kits’ instructions.


### 2.6. Apparent Digestibility Coefficients of Dry Matter and Protein

Freeze-dry diets and feces were firstly digested by nitric acid and hydrofluoric acid, and then, the content of yttrium oxide was measured by ICP-OES (Thermo Fisher 7200). The content of yttrium oxide in diets and feces was used in the calculation of apparent digestibility coefficient (ADC) of feeds.


### 2.7. Digestive Enzyme Activity

Activity of digestive enzymes (lipase, alpha-amylase, and trypsin) was analyzed by a spectrophotometer (Amersham Biosciences, Ultrospec 2100pro) according to the instructions of commercial reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).


### 2.8. Intestinal Morphology

The middle intestine was sliced into segments in 5 mm and dehydrated in the automatic dehydrator. After dehydration, tissues were embedded in paraffin and sliced in 6μm by a microtome (RM2235, LEICA). All the slices were stained with haematoxylin and eosin (H & E) using a linear slide stainer (A81500101, Thermo, UK) and enclosed by neutral balsam. Then, these slices were observed and photoed under different magnifications by an imaging microscope (CX31RTSF, Olympus, Japan). All the images were analyzed by the specialized software Imagine-Pro Plus 6.0; the villus height (VH), microvillus height (MH), and thickness of the intestinal wall (TIW) were recorded via the software.


### 2.9. Calculations and Statistical Analysis

The following parameters were calculated:(1)Survivalrate%=100×NtN0,SpecificgrowthrateSGR,%/day=lnWt−lnW0×100t,WeightgainrateWGR,%=100×Wt−W0W0,FeedintakeFI,%/day=100×Id/Wt+W0/2t,FeedefficiencyratioFER=Wt−W0Id,ProteinefficiencyratioPER=Wt−W0Id×P,ProteinretentionPR=Wt×Pt−W0×P0Id×P,ConditionfactorCF,g/cm3=100×bodyweightgbodylengthcm3,HepatosomaticindexHSI,%=100×liverweightgbodyweightg,ViscerosomaticindexVSI,%=100×visceraweightgbodyweightg,ApparentdigestibilitycoefficientADCofdrymatter%=100−dietaryY2O3fecalY2O3×100,ApparentdigestibilitycoefficientADCofnutrients%=100−dietaryY2Ο3fecalY2O3×fecalnutrientsdietarynutrients×100,whereW0 and Wt are the initial and final body weight, respectively. t is the duration of the feeding trial. Id is the feed intake as dry matter. P0, Pt, and P represent the protein content in initial fish body, final fish body, and diet, respectively. N0 and Nt are the initial and final number of fish, respectively.Data were analyzed by one-way ANOVA, Pearson’s correlation test, and linear regression analysis in software SPSS 17.0. In the analysis, data were initially examined for homogeneity of variances. Differences between means were tested by Tukey’s multiple range test. The level of significance was set atp<0.05, and results were presented as means±S.E.M. (standard error of the mean).


## 2.1. Experimental Diets

Seven isonitrogenous and isolipidic diets were formulated to contain approximately 520 g/kg crude protein and 110 g/kg crude lipid. These diets were divided into control group FM and six replacement groups FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6 (replacing 450 g/kg of fish meal with six different commercially available FSBM, which were collected from different firms in China through Qingdao Bio-ways Ingredients Bio-technology Co., Ltd.). Each diet containing FSBM was supplemented with coated L-methionine, L-isoleucine, and L-lysine to meet the requirements for juvenile turbot (Tables1 and 2). Yttrium oxide (Y2O3) was also added into each diet at the level of 1 g/kg as the indicator for dry matter and crude protein digestibility analysis. Ingredients were smashed into powder which could pass through 250 μm aperture mesh. All ingredients were thoroughly mixed with fish oil, and water was added in to produce moist dough. The moist dough was then pelleted by an experimental granulator (EL-220, Haiyang City Huatong Machinery Co., Ltd.) with 2 mm die and dried in a ventilated oven (CT-C-1, Jiang Yin Zhou Yuan Pharmaceutical Equipment Co., Ltd.) at 45°C for 10 h. All the pellets were stored at -20°C for use.Table 1
Formulation and proximate composition of experimental diets (g/kg dry matter)1.
IngredientsDiets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Brown fish meal1500.0275.0275.0275.0275.0275.0275.0Fermented soybean meal0.0300.4303.3293.2294.8293.0297.9Blood meal10.045.045.045.045.045.045.0Wheat gluten meal1134.092.090.090.090.090.092.0Wheat meal1244.5146.2142.4153.7150.9153.9146.6Fish oil45.062.062.063.064.062.064.0Soy lecithin20.020.020.020.020.020.020.0Vitamin premix315.015.015.015.015.015.015.0Vitamin C35.05.05.05.05.05.05.0Mineral premix415.015.015.015.015.015.015.0L-Methionine0.01.22.42.72.53.41.8L-Isoleucine0.01.11.10.70.90.80.8L-Lysine0.00.62.30.20.40.40.4Taurine5.05.05.05.05.05.05.0Choline chloride3.03.03.03.03.03.03.0Mildew preventive51.01.01.01.01.01.01.0Ethoxyquinoline0.50.50.50.50.50.50.5Ca(H2PO4)25.05.05.05.05.05.05.0Yttrium oxide1.01.01.01.01.01.01.0Sodium alginate1.01.01.01.01.01.01.0Attractants65.05.05.05.05.05.05.0Proximate compositionCrude protein525.5529.4522.2523.2525.1524.5529.0Crude lipid110.4109.7109.3112.1108.9109.6113.0Ash113.995.299.096.897.598.596.21Brown fish meal (g/kg dry matter): crude protein 724.7, crude lipid 98.4; blood meal (g/kg dry matter): crude protein 986.4, crude lipid 12.2; wheat gluten meal (g/kg dry matter): crude protein 791.5, crude lipid 19.3; wheat meal (g/kg dry matter): crude protein 139.3, crude lipid 24.7. These ingredients were provided by Great Seven Bio-tech (Qingdao, China). The commercial fermented soybean meal was collected by Qingdao Bio-ways Ingredients Bio-technology Co., Ltd. (Qingdao, China). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6. 3Vitamin premix (mg/kg diet): retinol acetate, 32; cholecalciferol, 5; alpha-tocopherol, 240; thiamin, 25; riboflavin, 45; pyridoxine HCl, 20; vitamin B12, 10; pantothenic acid, 60; folic acid, 20; niacin 200; biotin, 60; inositol, 800; microcrystalline cellulose, 13,473. Vitamin C was supplied in the form of vitamin C polyphosphate. 4Mineral premix (mg/kg diet): MgSO4·7H2O, 1200; CuSO4·5H2O, 10; FeSO4·H2O, 80; ZnSO4·H2O, 50; MnSO4·H2O, 45; CoCl2·6H2O, 50; Na2SeO3, 20; H2CaIO4, 60; zeolite powder, 13,485. 5Mildew preventive: calciumpropionate:boleticacid=1:1. 6Attractants: glycine betaine : DMPT : glycine : alanine : inosine-5-diphosphate trisodium salt=4:2:2:1:1.Table 2
Amino acid composition of experimental diets (g/kg dry matter)1.
Amino acidsDiets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Essential amino acidThreonine19.119.117.919.320.218.918.5Valine24.026.026.426.628.925.826.4Methionine12.211.713.013.513.714.111.8Isoleucine22.121.722.022.324.122.021.5Leucine36.639.339.441.043.940.139.1Phenylalanine22.725.225.926.728.225.224.1Lysine28.127.228.129.130.828.027.2Histidine14.114.815.015.516.715.014.7Arginine25.225.726.127.629.127.026.8Nonessential amino acidProline26.024.123.724.225.624.524.0Aspartic acid36.641.841.744.547.643.943.3Serine20.821.421.022.823.722.621.4Glutamic acid104.196.293.598.8106.097.594.9Glycine29.227.027.327.429.427.526.3Alanine30.130.429.330.232.429.929.0Cysteine5.35.25.45.76.35.85.4Tyrosine15.615.616.816.817.815.915.11Data are means of triplicate. No tryptophan was detected because of acid hydrolysis. 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6.


## 2.2. Experimental Fish and Procedure

Juvenile turbot were purchased from a commercial hatchery in Weihai, China. The feeding trial was conducted in LaiZhou MingBo Aquatic Co., Ltd. (Yantai, China). Turbot were fed with commercial diets for 14 days to acclimate to the experimental conditions. At the start of the feeding trial, all the turbot were fasted for 24 h and some of the fish were randomly weighed to estimate the average weight of the batch. Afterwards, fish of similar weight (6.99±0.02g) were selected and randomly assigned into 21 experimental fibreglass tanks (600 L capacity). Each diet was randomly assigned to triplicate tanks. Fish were fed twice daily (08:00 and 18:00) to apparent satiation for 10 weeks. After feeding, feces waste was cleaned up and seawater was also renewed. During the feeding trial, the water temperature ranged from 18 to 20°C, salinity ranged from 30 to 32, and dissolved oxygen maintained at approximately 6.0 mg/L. Meanwhile, the photoperiod was maintained on 12 h light : 12 h dark.


## 2.3. Sample Collection

At the start of formal growth experiment, twenty fish were randomly sampled from the remaining turbot for initial carcass protein analysis. From the seventh week of the growth experiment, feces samples were collected by siphoning after 8 h of feeding. All the collected feces samples were stored at -20°C for analysis. At the termination of the feeding trial, all the fish were fasted for 24 h and anesthetized with eugenol (1 : 12,000) to reduce the stress reaction before sampling. Then, the total number and weight of fish in each tank were first recorded. Six fish were randomly sampled from each tank to measure individual body weight, body length, visceral weight, and liver weight. Intact intestines were sampled from four fish per tank and frozen in liquid nitrogen immediately and then stored at -80°C for digestive enzyme analysis. Moreover, the middle intestine was sampled from three fish each tank for histology analysis. The sampled middle intestine (1 cm in length) were placed in 4% paraformaldehyde solution for fixation and then transferred to 75% ethanol after 24 h.


## 2.4. Biochemical Analysis

Proximate composition of ingredients, diets, and feces was analyzed according to published standards [25]. Moisture was determined by drying samples to a constant weight at 105°C for 24 h. Ash was measured gravimetrically by placing the samples in muffle furnace at 550°C for 16 h after ignition. Crude lipid was analyzed by petroleum ether extraction using Soxhlet (Buchi 36680, Switzerland). Crude protein was analyzed by the Kjeldahl method (Kjeltec™ 8400, FOSS, Sweden). Amino acids of ingredients and diets were analyzed by an amino acid analyzer (L-8900, Hitachi) after acid hydrolysis (6 N HCl for 24 h at 110°C). The content of water-soluble protein, trichloroacetic acid- (TCA-) soluble protein, and alkali- (KOH-) soluble protein was determined according to the methods described by Sarin [26] and Araba and Dale [27] with some modifications. Among them, the water-soluble protein in soybean meal was determined after three times of ultrapure water extraction in a shaker. For the determination of TCA-soluble protein, soybean meal was extracted by 15% trichloroacetic acid for 5 min, and supernatant was centrifuged (4000 r/min, 5 min) for protein determination. As to the analysis of alkali-soluble protein, soybean meal was extracted by 0.2% potassium hydroxide for 20 min in a magnetic stirrer; the supernatant used for protein determination was then obtained after centrifugation (2700 r/min, 10 min). The total calcium and phosphorus contents of these six FSBM were measured by an inductively coupled plasma-atomic emission spectrophotometer (ICP-OES, Thermo Fisher 7200).


## 2.5. Antinutritional Factor Analysis

The trypsin inhibitor, glycinin, andβ-conglycinin contents of FSBM were all analyzed by a microplate reader (SpectraMax i3, Molecular Devices) using competitive enzyme-linked immunosorbent assay (ELISA) kits (Beijing Longkefangzhou Bio-Engineering Technology Co., Ltd., China); all procedures performed in the analysis were in strict accordance with the kits’ instructions.


## 2.6. Apparent Digestibility Coefficients of Dry Matter and Protein

Freeze-dry diets and feces were firstly digested by nitric acid and hydrofluoric acid, and then, the content of yttrium oxide was measured by ICP-OES (Thermo Fisher 7200). The content of yttrium oxide in diets and feces was used in the calculation of apparent digestibility coefficient (ADC) of feeds.


## 2.7. Digestive Enzyme Activity

Activity of digestive enzymes (lipase, alpha-amylase, and trypsin) was analyzed by a spectrophotometer (Amersham Biosciences, Ultrospec 2100pro) according to the instructions of commercial reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).


## 2.8. Intestinal Morphology

The middle intestine was sliced into segments in 5 mm and dehydrated in the automatic dehydrator. After dehydration, tissues were embedded in paraffin and sliced in 6μm by a microtome (RM2235, LEICA). All the slices were stained with haematoxylin and eosin (H & E) using a linear slide stainer (A81500101, Thermo, UK) and enclosed by neutral balsam. Then, these slices were observed and photoed under different magnifications by an imaging microscope (CX31RTSF, Olympus, Japan). All the images were analyzed by the specialized software Imagine-Pro Plus 6.0; the villus height (VH), microvillus height (MH), and thickness of the intestinal wall (TIW) were recorded via the software.


## 2.9. Calculations and Statistical Analysis

The following parameters were calculated:(1)Survivalrate%=100×NtN0,SpecificgrowthrateSGR,%/day=lnWt−lnW0×100t,WeightgainrateWGR,%=100×Wt−W0W0,FeedintakeFI,%/day=100×Id/Wt+W0/2t,FeedefficiencyratioFER=Wt−W0Id,ProteinefficiencyratioPER=Wt−W0Id×P,ProteinretentionPR=Wt×Pt−W0×P0Id×P,ConditionfactorCF,g/cm3=100×bodyweightgbodylengthcm3,HepatosomaticindexHSI,%=100×liverweightgbodyweightg,ViscerosomaticindexVSI,%=100×visceraweightgbodyweightg,ApparentdigestibilitycoefficientADCofdrymatter%=100−dietaryY2O3fecalY2O3×100,ApparentdigestibilitycoefficientADCofnutrients%=100−dietaryY2Ο3fecalY2O3×fecalnutrientsdietarynutrients×100,whereW0 and Wt are the initial and final body weight, respectively. t is the duration of the feeding trial. Id is the feed intake as dry matter. P0, Pt, and P represent the protein content in initial fish body, final fish body, and diet, respectively. N0 and Nt are the initial and final number of fish, respectively.Data were analyzed by one-way ANOVA, Pearson’s correlation test, and linear regression analysis in software SPSS 17.0. In the analysis, data were initially examined for homogeneity of variances. Differences between means were tested by Tukey’s multiple range test. The level of significance was set atp<0.05, and results were presented as means±S.E.M. (standard error of the mean).


## 3. Results

### 3.1. Nutritional Composition and Antinutritional Factor Content of Fermented Soybean Meal

The content of nutrients and ANFs in FSBM, including crude lipid, crude protein, water-soluble protein, TCA-soluble protein, alkali- (KOH-) soluble protein, ash, total calcium, total phosphorus, amino acid profile, trypsin inhibitor, glycinin, andβ-conglycinin (Tables 3 and 4), was all analyzed. The lipid content of the six tested FSBM ranged from 17.1 to 25.5 g/kg, with values in decreasing order of FSBM2, FSBM1, FSBM5, FSBM3, FSBM4, and FSBM6. Besides, the protein content of FSBM3 and FSBM5 (555.6 and 556.0 g/kg) was significantly higher than that of other FSBM (ranging from 537.1 to 552.6 g/kg) (p<0.05). As to the water-soluble protein and TCA-soluble protein, they were significantly higher in FSBM4 and FSBM3 than in other FSBM (p<0.05). The alkali- (KOH-) soluble protein in FSBM1 and FSBM2 was significantly higher than that in others (p<0.05). In terms of ANFs, significantly higher levels of trypsin inhibitor and glycinin were observed in FSBM1 and FSBM2, while the β-conglycinin content of FSBM3 and FSBM5 was significantly higher than that of other FSBM (p<0.05), whereas FSBM4 showed significantly lower levels of trypsin inhibitor and β-conglycinin (p<0.05).Table 3
Nutritional composition and antinutritional factor content of fermented soybean meal (dry matter basic,means±S.E.M.)1.
Fermented soybean meal2FSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Crude lipid (g/kg)25.2±0.1a25.5±0.1a21.8±0.1c18.6±0.1d24.5±0.1b17.1±0.1eCrude protein (g/kg)542.3±1.2d537.1±0.2e555.6±0.9a552.6±0.3b556.0±0.8a546.8±1.1cWater-soluble protein (g/kg)357.7±2.2d47.9±1.0e82.7±0.8b91.3±0.1a68.1±0.8c49.9±1.0eTCA-soluble protein (%)42.9±0.2d2.1±0.1e5.2±0.1b5.8±0.3a4.2±0.1c2.1±0.1eAlkali- (KOH-) soluble protein (%)585.5±0.2a86.5±0.2a82.6±0.8b75.8±0.3d77.8±0.1c75.9±0.2dAsh (g/kg)70.3±0.3c83.1±0.7ab84.9±1.5a80.3±0.6b83.5±1.2ab82.6±1.0abTotal calcium (g/kg)3.6±0.6b3.5±0.2b4.2±1.0a4.0±0.6a4.2±0.5a4.1±0.9aTotal phosphorus (g/kg)7.5±0.5c7.7±0.1c8.3±0.3ab8.5±1.2a8.2±1.0b8.2±0.6bTrypsin inhibitor (mg/g)3.4±0.1a3.4±0.2a1.5±0.5c1.2±0.2c2.6±0.1b3.3±0.1aGlycinin (mg/g)39.4±0.3a40.9±0.1a32.3±0.4c35.6±0.3b15.6±0.2e22.6±0.2dβ-Conglycinin (mg/g)38.6±0.2d47.6±0.1c60.7±0.1a15.5±0.1e59.5±0.2a51.9±0.1b1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2These six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6 to protect corporate privacy. 3Protein extracted by ultrapure water for three times. 4Ratio of the protein extracted by 15% trichloroacetic acid to the total crude protein. 5Ratio of the protein extracted by 0.2% potassium hydroxide to the total crude protein.Table 4
Amino acid profiles of fermented soybean meal (g/kg dry matter,means±S.E.M.)1.
Amino acidsFermented soybean meal2FSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Essential amino acidsThreonine24.1±0.4a20.9±0.1b21.4±0.5b22.2±0.1b21.9±0.4b22.3±0.1bValine24.5±0.124.8±0.223.5±1.123.4±0.123.8±0.623.9±0.1Methionine4.9±1.4a5.1±0.3a4.7±0.7a3.8±1.1b3.7±0.5b3.7±1.2bIsoleucine21.9±0.3b24.2±0.5a22.5±0.9ab22.7±0.3ab22.6±0.3ab22.7±0.3abLeucine39.7±0.840.4±0.340.2±1.141.6±0.341.0±0.841.5±0.3Phenylalanine25.1±0.5c26.3±0.1bc27.0±0.7ab28.7±0.8a27.7±0.5ab27.6±0.1abLysine26.7±0.727.8±0.127.6±0.828.0±0.227.8±0.627.7±0.1Histidine12.2±0.4b12.6±0.3ab12.9±0.4ab13.5±0.1a13.1±0.3ab13.2±0.2abArginine34.3±0.7c36.2±0.1bc36.7±1.2b35.7±0.2bc36.9±0.7b37.7±0.2aNonessential amino acidsProline22.0±1.5b23.7±0.5ab25.6±0.7a26.5±0.2a25.6±0.5a26.4±0.1aAspartic acid57.5±1.2b59.0±0.3ab59.8±1.4ab61.6±0.3a59.7±1.2ab61.1±0.2aSerine26.0±0.5b26.2±0.3b27.4±0.5ab28.9±0.3a29.0±0.7a28.8±0.1aGlutamic acid97.2±1.7b103.3±0.4a104.3±2.5a106.1±0.5a103.2±2.2a105.8±0.2aGlycine24.2±0.7a22.4±0.1b22.1±0.6b23.2±0.1ab23.2±0.5ab22.9±0.1abAlanine28.2±0.5a22.6±0.1c24.1±0.6b24.7±0.1b24.9±0.5b24.5±0.1bCysteine5.6±0.5b6.4±0.1a6.4±0.2a6.6±0.2a6.5±0.1a6.5±0.3aTyrosine16.5±0.7b17.5±0.1b18.0±0.6a19.1±0.5a18.0±0.2a18.1±0.2a1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2These six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6 to protect corporate privacy.No significant differences were observed in the content of valine (Val), leucine (Leu), and lysine (Lys) among the six different FSBM (p>0.05), while FSBM1 showed significantly lower levels of isoleucine (Ile), phenylalanine (Phe), histidine (His), and arginine (Arg) than the rest (p<0.05). Meanwhile, the content of Phe in FSBM4 was significantly higher than that of FSBM1 and FSBM2 (p<0.05), and the content of His showed similar pattern as that of Phe.


### 3.2. Growth Performance and Feed Utilization

The survival rate of fish ranged from 97.78 to 100%, but no significant differences were observed among dietary treatments (p>0.05) (Table 5). FI revealed no significant differences among turbot fed diets with FSBM2, FSBM3, FSBM5, and FM (p>0.05), while fish fed the diet with FSBM1 showed significantly lower FI than the other groups (p<0.05). The SGR was significantly higher in FSBM3 and FSBM4 than in other replacement groups (p<0.05), but significantly lower than in the control group (p<0.05). No significant differences were detected in FER and PER among groups FSBM3, FSBM4, and FM (p>0.05), but their values were significantly lower in groups FSBM1 and FSBM2 than in others (p<0.05). PR showed a similar pattern as FER and PER. HSI and CF were significantly lower in group FSBM1 than in others (p<0.05), while VSI was significantly higher in groups FSBM1 and FSBM2 as compared with the control (p<0.05).Table 5
Growth parameters and feed utilization of juvenile turbot (Scophthalmus maximus L.) fed the experimental diets (means±S.E.M.)1.
Diets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6IBW (g)36.99±0.026.99±0.026.99±0.026.99±0.026.99±0.026.99±0.026.99±0.02FBW (g)330.80±1.08a15.04±0.88d16.06±1.25d24.59±0.65b24.68±0.31b20.20±0.72c19.08±0.27cWGR (%)3340.06±15.41a114.85±12.53d129.47±17.81d251.24±9.24b252.60±4.43b188.62±10.35c172.53±3.84cFI (%/day)31.50±0.06a1.10±0.02c1.36±0.08ab1.39±0.01ab1.26±0.03b1.44±0.04a1.28±0.04bSGR (%/day)32.12±0.05a1.09±0.08d1.18±0.11d1.79±0.04b1.80±0.02b1.51±0.05c1.43±0.02cSurvival rate (%)100.00±0.0098.90±0.9197.80±0.91100.00±0.00100.00±0.0097.78±2.23100.00±0.00FER31.27±0.07a0.99±0.06cd0.87±0.03d1.20±0.03ab1.33±0.02a1.00±0.01cd1.08±0.03bcPER32.41±0.14a1.86±0.12cd1.66±0.06d2.29±0.06ab2.54±0.04a1.91±0.01cd2.07±0.06bcPR30.34±0.02a0.25±0.02c0.21±0.01d0.30±0.01b0.36±0.01a0.26±0.01c0.27±0.01bcHSI (%)30.84±0.02a0.53±0.06b0.84±0.04a0.77±0.02a0.79±0.03a0.80±0.04a0.79±0.04aVSI (%)33.89±0.13c4.50±0.08a4.35±0.15ab4.04±0.17bc3.93±0.07c4.22±0.12abc4.03±0.07bcCF (g/cm3)33.52±0.03a3.18±0.09b3.52±0.09a3.67±0.10a3.61±0.05a3.51±0.11a3.56±0.05a1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6. 3IBW: initial body weight; FBW: final body weight; WGR: weight gain rate; FI: feed intake; SGR: specific growth rate; FER: feed efficiency ratio; PER: protein efficiency ratio; PR: protein retention; HSI: hepatosomatic index; VSI: viscerosomatic index; CF: condition factor.


### 3.3. Apparent Digestibility Coefficients of Dry Matter and Protein

The apparent digestibility coefficient (ADC) of dry matter ranged from 44.65 to 61.46% (Table6) and its values decreased in the order of FSBM4, FM, FSBM6, FSBM3, FSBM5, FSBM1, and FSBM2, where no significant difference was observed between groups FSBM4 and FM (p>0.05). The ADC of protein ranged from 83.34 to 92.07%, which decreased in the order of FM, FSBM4, FSBM6, FSBM3, FSBM5, FSBM2, and FSBM1.Table 6
Apparent digestibility coefficients (%, ADC) of dry matter and protein for the experimental diets (means±S.E.M.)1.
Diets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6ADC of dry matter59.17±0.90a44.66±1.44d44.65±1.09d49.62±0.82c61.46±2.02a48.46±0.86c53.52±0.63bADC of protein92.07±0.17a83.34±0.43e84.86±0.30d86.93±0.21c90.86±0.48b86.81±0.22c87.53±0.17c1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6.


### 3.4. Pearson’s Correlation Test and Linear Regression Analysis

The correlation analysis showed that SGR and PER were both positively correlated with certain FSBM components, such as water-soluble protein, TCA-soluble protein, and total phosphorus (Table7) (p<0.05), while the PR was only positively correlated with the content of water-soluble protein and TCA-soluble protein (p<0.05). Besides, the trypsin inhibitor content of FSBM showed negative correlation with SGR, PER, and PR. In addition, SGR, PER, and PR were all positively correlated with the ADC of dry matter and protein (Table 8). Based on Pearson’s correlation test, the corresponding linear regression analysis was also performed (Tables 9 and 10).Table 7
Pearson’s correlation test between growth parameters and fermented soybean meal components.
Growth parametersFermented soybean meal componentsr2p2SGR1Crude lipid-0.583ns3Crude protein0.845<0.05Ash0.562nsWater-soluble protein0.863<0.05TCA-soluble protein10.856<0.05Total calcium0.846<0.05Total phosphorus0.961<0.05Trypsin inhibitor-0.928<0.05Glycinin-0.295nsβ-Conglycinin-0.108nsPER1Crude lipid-0.725nsCrude protein0.676nsAsh0.171nsWater-soluble protein0.864<0.05TCA-soluble protein0.824<0.05Total calcium0.607nsTotal phosphorus0.824<0.05Trypsin inhibitor-0.886<0.05Glycinin-0.056nsβ-Conglycinin-0.462nsPR1Crude lipid-0.673nsCrude protein0.674nsAsh0.086nsWater-soluble protein0.885<0.05TCA-soluble protein0.849<0.05Total calcium0.552nsTotal phosphorus0.805nsTrypsin inhibitor-0.884<0.05Glycinin-0.061nsβ-Conglycinin-0.554ns1SGR: specific growth rate; PER: protein efficiency ratio; PR: protein retention; TCA-soluble protein: ratio of the protein extracted by 15% trichloroacetic acid to the total crude protein. 2r: correlation index; p: test statistic (significant, p<0.05). 3ns: not significant.Table 8
Pearson’s correlation test between growth parameters and apparent digestibility coefficient (ADC) for feed.
Growth parametersApparent digestibility coefficient (ADC)r2p2SGR1ADC of dry matter0.812<0.05ADC of protein0.915<0.05PER1ADC of dry matter0.901<0.05ADC of protein0.857<0.05PR1ADC of dry matter0.920<0.05ADC of protein0.878<0.051SGR: specific growth rate; PER: protein efficiency ratio; PR: protein retention. 2r: correlation index; p: test statistic (significant, p<0.05).Table 9
Linear regression analysis between growth parameters and fermented soybean meal components.
Growth parametersFermented soybean meal componentsLinear regression line1R2p3SGR2Crude lipid0.34ns3Crude proteiny=0.328x−16.5300.71<0.05Ash0.32nsWater-soluble proteiny=0.145x+0.5080.75<0.05TCA-soluble protein2y=0.159x+0.8750.73<0.05Total calciumy=8.794x−2.0040.72<0.05Total phosphorusy=7.510x−4.6020.92<0.05Trypsin inhibitory=−0.274x+2.1670.86<0.05Glycinin0.09nsβ-Conglycinin0.01nsPER2Crude lipid0.53nsCrude protein0.46nsAsh0.03nsWater-soluble proteiny=1.55x+1.0290.75<0.05TCA-soluble proteiny=0.164x+1.4460.68<0.05Total calcium0.37nsTotal phosphorusy=6.884x−3.5070.68<0.05Trypsin inhibitory=−0.280x+2.7700.79<0.05Glycinin0.01nsβ-Conglycinin0.21nsPR2Crude lipid0.45nsCrude protein0.45nsAsh0.01nsWater-soluble proteiny=0.025x+0.1070.78<0.05TCA-soluble proteiny=0.027x+0.1750.72<0.05Total calcium0.31nsTotal phosphorus0.65nsTrypsin inhibitory=−0.045x+0.3890.78<0.05Glycinin0.01nsβ-Conglycinin0.31ns1Linear regression lines (where y is the growth parameters and x is the fermented soybean meal components). 2SGR: specific growth rate; PER: protein efficiency ratio; PR: protein retention; TCA-soluble protein: ratio of the protein extracted by 15% trichloroacetic acid to the total crude protein. 3p: test statistic (significant, p<0.05); ns: not significant.Table 10
Linear regression analysis between growth parameters and apparent digestibility coefficient (ADC) for feed1.
Growth performance/protein utilizationApparent digestibility coefficient (ADC)Linear regression line1R2pSGR2ADC of dry mattery=0.045x−0.7410.659<0.05ADC of proteiny=0.043x−0.1200.812<0.05PER2ADC of dry mattery=0.007x−0.0890.846<0.05ADC of proteiny=0.109x−7.9500.836<0.05PR2ADC of dry mattery=0.089x−5.6530.734<0.05ADC of proteiny=0.015x−1.0220.771<0.051Linear regression lines (where y is the growth parameters and x is the ADC of dry matter and protein). 2SGR: specific growth rate; PER: protein efficiency ratio; PR: protein retention.


### 3.5. Activity of Digestive Enzymes

The trypsin activity of group FSBM6 was significantly lower than that of groups FSBM1 and FM (p<0.05) (Table 11). As to the alpha-amylase, there were no significant differences among fish fed with diets FSBM1, FSBM2, and FM (p>0.05). Lipase showed significantly higher activity in group FSBM2 than in the control group (p<0.05), but no significant differences were observed in lipase activity among groups FSBM1, FSBM3, FSBM5, FSBM6, and FM (p>0.05).Table 11
Digestive enzyme activity of juvenile turbot (Scophthalmus maximus L.) fed the experimental diets (means±S.E.M.)1.
Diets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Trypsin (U/μg prot)24.62±2.85a18.59±2.19b14.09±0.21bc14.14±2.18bc14.53±1.28bc13.29±2.17bc10.91±0.94cAlpha-amylase (U/mg prot)0.34±0.02a0.28±0.02a0.28±0.05a0.17±0.10b0.18±0.05b0.18±0.02b0.15±0.04bLipase (U/g prot)1.58±0.34c2.23±0.29bc3.40±0.47a2.04±0.13c3.13±0.94ab2.61±0.28abc2.25±0.15bc1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6.


### 3.6. Intestinal Morphology

The villus height (VH) of group FSBM4 was significantly higher than that of groups FSBM1 and FSBM2 (p<0.05). As to microvillus height (MH) and thickness of the intestinal wall (TIW), they showed decreasing order by groups FM, FSBM4, FSBM6, FSBM3, FSBM5, FSBM2, and FSBM1 (Table 12 and Figure 1), but no significant differences were observed in MH between groups FSBM4 and FM (p>0.05).Table 12
Middle intestinal histology parameters of juvenile turbot (Scophthalmus maximus L.) fed the experimental diets (means±S.E.M.)1.
Diets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6VH (μm)3847.75±36.11ab607.72±23.48c645.94±24.76c714.26±37.80ab751.38±44.59ab874.48±76.68a818.37±47.61abTIW (μm)3158.38±3.78a92.43±2.37d101.89±10.63cd122.49±10.10bc128.67±4.19b115.88±7.26bc135.85±4.19bMH (μm)34.73±0.23a3.36±0.17c3.62±0.05bc3.87±0.06bc4.32±0.46ab3.82±0.29bc3.88±0.05bc1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6. 3VH: villus height; TIW: thickness of the intestinal wall; MH: microvillus height.Figure 1
Representative intestine sections of juvenile turbot (Scophthalmus maximus L.) fed with (a) fish-meal-based diet FM and diets containing (b) FSBM1, (c) FSBM2, (d) FSBM3, (e) FSBM4, (f) FSBM5, and (g) FSBM6, all the sections were stained by haematoxylin and eosin. Scale bars, 100 μm.
(a)(b)(c)(d)(e)(f)(g)


## 3.1. Nutritional Composition and Antinutritional Factor Content of Fermented Soybean Meal

The content of nutrients and ANFs in FSBM, including crude lipid, crude protein, water-soluble protein, TCA-soluble protein, alkali- (KOH-) soluble protein, ash, total calcium, total phosphorus, amino acid profile, trypsin inhibitor, glycinin, andβ-conglycinin (Tables 3 and 4), was all analyzed. The lipid content of the six tested FSBM ranged from 17.1 to 25.5 g/kg, with values in decreasing order of FSBM2, FSBM1, FSBM5, FSBM3, FSBM4, and FSBM6. Besides, the protein content of FSBM3 and FSBM5 (555.6 and 556.0 g/kg) was significantly higher than that of other FSBM (ranging from 537.1 to 552.6 g/kg) (p<0.05). As to the water-soluble protein and TCA-soluble protein, they were significantly higher in FSBM4 and FSBM3 than in other FSBM (p<0.05). The alkali- (KOH-) soluble protein in FSBM1 and FSBM2 was significantly higher than that in others (p<0.05). In terms of ANFs, significantly higher levels of trypsin inhibitor and glycinin were observed in FSBM1 and FSBM2, while the β-conglycinin content of FSBM3 and FSBM5 was significantly higher than that of other FSBM (p<0.05), whereas FSBM4 showed significantly lower levels of trypsin inhibitor and β-conglycinin (p<0.05).Table 3
Nutritional composition and antinutritional factor content of fermented soybean meal (dry matter basic,means±S.E.M.)1.
Fermented soybean meal2FSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Crude lipid (g/kg)25.2±0.1a25.5±0.1a21.8±0.1c18.6±0.1d24.5±0.1b17.1±0.1eCrude protein (g/kg)542.3±1.2d537.1±0.2e555.6±0.9a552.6±0.3b556.0±0.8a546.8±1.1cWater-soluble protein (g/kg)357.7±2.2d47.9±1.0e82.7±0.8b91.3±0.1a68.1±0.8c49.9±1.0eTCA-soluble protein (%)42.9±0.2d2.1±0.1e5.2±0.1b5.8±0.3a4.2±0.1c2.1±0.1eAlkali- (KOH-) soluble protein (%)585.5±0.2a86.5±0.2a82.6±0.8b75.8±0.3d77.8±0.1c75.9±0.2dAsh (g/kg)70.3±0.3c83.1±0.7ab84.9±1.5a80.3±0.6b83.5±1.2ab82.6±1.0abTotal calcium (g/kg)3.6±0.6b3.5±0.2b4.2±1.0a4.0±0.6a4.2±0.5a4.1±0.9aTotal phosphorus (g/kg)7.5±0.5c7.7±0.1c8.3±0.3ab8.5±1.2a8.2±1.0b8.2±0.6bTrypsin inhibitor (mg/g)3.4±0.1a3.4±0.2a1.5±0.5c1.2±0.2c2.6±0.1b3.3±0.1aGlycinin (mg/g)39.4±0.3a40.9±0.1a32.3±0.4c35.6±0.3b15.6±0.2e22.6±0.2dβ-Conglycinin (mg/g)38.6±0.2d47.6±0.1c60.7±0.1a15.5±0.1e59.5±0.2a51.9±0.1b1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2These six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6 to protect corporate privacy. 3Protein extracted by ultrapure water for three times. 4Ratio of the protein extracted by 15% trichloroacetic acid to the total crude protein. 5Ratio of the protein extracted by 0.2% potassium hydroxide to the total crude protein.Table 4
Amino acid profiles of fermented soybean meal (g/kg dry matter,means±S.E.M.)1.
Amino acidsFermented soybean meal2FSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Essential amino acidsThreonine24.1±0.4a20.9±0.1b21.4±0.5b22.2±0.1b21.9±0.4b22.3±0.1bValine24.5±0.124.8±0.223.5±1.123.4±0.123.8±0.623.9±0.1Methionine4.9±1.4a5.1±0.3a4.7±0.7a3.8±1.1b3.7±0.5b3.7±1.2bIsoleucine21.9±0.3b24.2±0.5a22.5±0.9ab22.7±0.3ab22.6±0.3ab22.7±0.3abLeucine39.7±0.840.4±0.340.2±1.141.6±0.341.0±0.841.5±0.3Phenylalanine25.1±0.5c26.3±0.1bc27.0±0.7ab28.7±0.8a27.7±0.5ab27.6±0.1abLysine26.7±0.727.8±0.127.6±0.828.0±0.227.8±0.627.7±0.1Histidine12.2±0.4b12.6±0.3ab12.9±0.4ab13.5±0.1a13.1±0.3ab13.2±0.2abArginine34.3±0.7c36.2±0.1bc36.7±1.2b35.7±0.2bc36.9±0.7b37.7±0.2aNonessential amino acidsProline22.0±1.5b23.7±0.5ab25.6±0.7a26.5±0.2a25.6±0.5a26.4±0.1aAspartic acid57.5±1.2b59.0±0.3ab59.8±1.4ab61.6±0.3a59.7±1.2ab61.1±0.2aSerine26.0±0.5b26.2±0.3b27.4±0.5ab28.9±0.3a29.0±0.7a28.8±0.1aGlutamic acid97.2±1.7b103.3±0.4a104.3±2.5a106.1±0.5a103.2±2.2a105.8±0.2aGlycine24.2±0.7a22.4±0.1b22.1±0.6b23.2±0.1ab23.2±0.5ab22.9±0.1abAlanine28.2±0.5a22.6±0.1c24.1±0.6b24.7±0.1b24.9±0.5b24.5±0.1bCysteine5.6±0.5b6.4±0.1a6.4±0.2a6.6±0.2a6.5±0.1a6.5±0.3aTyrosine16.5±0.7b17.5±0.1b18.0±0.6a19.1±0.5a18.0±0.2a18.1±0.2a1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2These six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6 to protect corporate privacy.No significant differences were observed in the content of valine (Val), leucine (Leu), and lysine (Lys) among the six different FSBM (p>0.05), while FSBM1 showed significantly lower levels of isoleucine (Ile), phenylalanine (Phe), histidine (His), and arginine (Arg) than the rest (p<0.05). Meanwhile, the content of Phe in FSBM4 was significantly higher than that of FSBM1 and FSBM2 (p<0.05), and the content of His showed similar pattern as that of Phe.


## 3.2. Growth Performance and Feed Utilization

The survival rate of fish ranged from 97.78 to 100%, but no significant differences were observed among dietary treatments (p>0.05) (Table 5). FI revealed no significant differences among turbot fed diets with FSBM2, FSBM3, FSBM5, and FM (p>0.05), while fish fed the diet with FSBM1 showed significantly lower FI than the other groups (p<0.05). The SGR was significantly higher in FSBM3 and FSBM4 than in other replacement groups (p<0.05), but significantly lower than in the control group (p<0.05). No significant differences were detected in FER and PER among groups FSBM3, FSBM4, and FM (p>0.05), but their values were significantly lower in groups FSBM1 and FSBM2 than in others (p<0.05). PR showed a similar pattern as FER and PER. HSI and CF were significantly lower in group FSBM1 than in others (p<0.05), while VSI was significantly higher in groups FSBM1 and FSBM2 as compared with the control (p<0.05).Table 5
Growth parameters and feed utilization of juvenile turbot (Scophthalmus maximus L.) fed the experimental diets (means±S.E.M.)1.
Diets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6IBW (g)36.99±0.026.99±0.026.99±0.026.99±0.026.99±0.026.99±0.026.99±0.02FBW (g)330.80±1.08a15.04±0.88d16.06±1.25d24.59±0.65b24.68±0.31b20.20±0.72c19.08±0.27cWGR (%)3340.06±15.41a114.85±12.53d129.47±17.81d251.24±9.24b252.60±4.43b188.62±10.35c172.53±3.84cFI (%/day)31.50±0.06a1.10±0.02c1.36±0.08ab1.39±0.01ab1.26±0.03b1.44±0.04a1.28±0.04bSGR (%/day)32.12±0.05a1.09±0.08d1.18±0.11d1.79±0.04b1.80±0.02b1.51±0.05c1.43±0.02cSurvival rate (%)100.00±0.0098.90±0.9197.80±0.91100.00±0.00100.00±0.0097.78±2.23100.00±0.00FER31.27±0.07a0.99±0.06cd0.87±0.03d1.20±0.03ab1.33±0.02a1.00±0.01cd1.08±0.03bcPER32.41±0.14a1.86±0.12cd1.66±0.06d2.29±0.06ab2.54±0.04a1.91±0.01cd2.07±0.06bcPR30.34±0.02a0.25±0.02c0.21±0.01d0.30±0.01b0.36±0.01a0.26±0.01c0.27±0.01bcHSI (%)30.84±0.02a0.53±0.06b0.84±0.04a0.77±0.02a0.79±0.03a0.80±0.04a0.79±0.04aVSI (%)33.89±0.13c4.50±0.08a4.35±0.15ab4.04±0.17bc3.93±0.07c4.22±0.12abc4.03±0.07bcCF (g/cm3)33.52±0.03a3.18±0.09b3.52±0.09a3.67±0.10a3.61±0.05a3.51±0.11a3.56±0.05a1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6. 3IBW: initial body weight; FBW: final body weight; WGR: weight gain rate; FI: feed intake; SGR: specific growth rate; FER: feed efficiency ratio; PER: protein efficiency ratio; PR: protein retention; HSI: hepatosomatic index; VSI: viscerosomatic index; CF: condition factor.


## 3.3. Apparent Digestibility Coefficients of Dry Matter and Protein

The apparent digestibility coefficient (ADC) of dry matter ranged from 44.65 to 61.46% (Table6) and its values decreased in the order of FSBM4, FM, FSBM6, FSBM3, FSBM5, FSBM1, and FSBM2, where no significant difference was observed between groups FSBM4 and FM (p>0.05). The ADC of protein ranged from 83.34 to 92.07%, which decreased in the order of FM, FSBM4, FSBM6, FSBM3, FSBM5, FSBM2, and FSBM1.Table 6
Apparent digestibility coefficients (%, ADC) of dry matter and protein for the experimental diets (means±S.E.M.)1.
Diets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6ADC of dry matter59.17±0.90a44.66±1.44d44.65±1.09d49.62±0.82c61.46±2.02a48.46±0.86c53.52±0.63bADC of protein92.07±0.17a83.34±0.43e84.86±0.30d86.93±0.21c90.86±0.48b86.81±0.22c87.53±0.17c1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6.


## 3.4. Pearson’s Correlation Test and Linear Regression Analysis

The correlation analysis showed that SGR and PER were both positively correlated with certain FSBM components, such as water-soluble protein, TCA-soluble protein, and total phosphorus (Table7) (p<0.05), while the PR was only positively correlated with the content of water-soluble protein and TCA-soluble protein (p<0.05). Besides, the trypsin inhibitor content of FSBM showed negative correlation with SGR, PER, and PR. In addition, SGR, PER, and PR were all positively correlated with the ADC of dry matter and protein (Table 8). Based on Pearson’s correlation test, the corresponding linear regression analysis was also performed (Tables 9 and 10).Table 7
Pearson’s correlation test between growth parameters and fermented soybean meal components.
Growth parametersFermented soybean meal componentsr2p2SGR1Crude lipid-0.583ns3Crude protein0.845<0.05Ash0.562nsWater-soluble protein0.863<0.05TCA-soluble protein10.856<0.05Total calcium0.846<0.05Total phosphorus0.961<0.05Trypsin inhibitor-0.928<0.05Glycinin-0.295nsβ-Conglycinin-0.108nsPER1Crude lipid-0.725nsCrude protein0.676nsAsh0.171nsWater-soluble protein0.864<0.05TCA-soluble protein0.824<0.05Total calcium0.607nsTotal phosphorus0.824<0.05Trypsin inhibitor-0.886<0.05Glycinin-0.056nsβ-Conglycinin-0.462nsPR1Crude lipid-0.673nsCrude protein0.674nsAsh0.086nsWater-soluble protein0.885<0.05TCA-soluble protein0.849<0.05Total calcium0.552nsTotal phosphorus0.805nsTrypsin inhibitor-0.884<0.05Glycinin-0.061nsβ-Conglycinin-0.554ns1SGR: specific growth rate; PER: protein efficiency ratio; PR: protein retention; TCA-soluble protein: ratio of the protein extracted by 15% trichloroacetic acid to the total crude protein. 2r: correlation index; p: test statistic (significant, p<0.05). 3ns: not significant.Table 8
Pearson’s correlation test between growth parameters and apparent digestibility coefficient (ADC) for feed.
Growth parametersApparent digestibility coefficient (ADC)r2p2SGR1ADC of dry matter0.812<0.05ADC of protein0.915<0.05PER1ADC of dry matter0.901<0.05ADC of protein0.857<0.05PR1ADC of dry matter0.920<0.05ADC of protein0.878<0.051SGR: specific growth rate; PER: protein efficiency ratio; PR: protein retention. 2r: correlation index; p: test statistic (significant, p<0.05).Table 9
Linear regression analysis between growth parameters and fermented soybean meal components.
Growth parametersFermented soybean meal componentsLinear regression line1R2p3SGR2Crude lipid0.34ns3Crude proteiny=0.328x−16.5300.71<0.05Ash0.32nsWater-soluble proteiny=0.145x+0.5080.75<0.05TCA-soluble protein2y=0.159x+0.8750.73<0.05Total calciumy=8.794x−2.0040.72<0.05Total phosphorusy=7.510x−4.6020.92<0.05Trypsin inhibitory=−0.274x+2.1670.86<0.05Glycinin0.09nsβ-Conglycinin0.01nsPER2Crude lipid0.53nsCrude protein0.46nsAsh0.03nsWater-soluble proteiny=1.55x+1.0290.75<0.05TCA-soluble proteiny=0.164x+1.4460.68<0.05Total calcium0.37nsTotal phosphorusy=6.884x−3.5070.68<0.05Trypsin inhibitory=−0.280x+2.7700.79<0.05Glycinin0.01nsβ-Conglycinin0.21nsPR2Crude lipid0.45nsCrude protein0.45nsAsh0.01nsWater-soluble proteiny=0.025x+0.1070.78<0.05TCA-soluble proteiny=0.027x+0.1750.72<0.05Total calcium0.31nsTotal phosphorus0.65nsTrypsin inhibitory=−0.045x+0.3890.78<0.05Glycinin0.01nsβ-Conglycinin0.31ns1Linear regression lines (where y is the growth parameters and x is the fermented soybean meal components). 2SGR: specific growth rate; PER: protein efficiency ratio; PR: protein retention; TCA-soluble protein: ratio of the protein extracted by 15% trichloroacetic acid to the total crude protein. 3p: test statistic (significant, p<0.05); ns: not significant.Table 10
Linear regression analysis between growth parameters and apparent digestibility coefficient (ADC) for feed1.
Growth performance/protein utilizationApparent digestibility coefficient (ADC)Linear regression line1R2pSGR2ADC of dry mattery=0.045x−0.7410.659<0.05ADC of proteiny=0.043x−0.1200.812<0.05PER2ADC of dry mattery=0.007x−0.0890.846<0.05ADC of proteiny=0.109x−7.9500.836<0.05PR2ADC of dry mattery=0.089x−5.6530.734<0.05ADC of proteiny=0.015x−1.0220.771<0.051Linear regression lines (where y is the growth parameters and x is the ADC of dry matter and protein). 2SGR: specific growth rate; PER: protein efficiency ratio; PR: protein retention.


## 3.5. Activity of Digestive Enzymes

The trypsin activity of group FSBM6 was significantly lower than that of groups FSBM1 and FM (p<0.05) (Table 11). As to the alpha-amylase, there were no significant differences among fish fed with diets FSBM1, FSBM2, and FM (p>0.05). Lipase showed significantly higher activity in group FSBM2 than in the control group (p<0.05), but no significant differences were observed in lipase activity among groups FSBM1, FSBM3, FSBM5, FSBM6, and FM (p>0.05).Table 11
Digestive enzyme activity of juvenile turbot (Scophthalmus maximus L.) fed the experimental diets (means±S.E.M.)1.
Diets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6Trypsin (U/μg prot)24.62±2.85a18.59±2.19b14.09±0.21bc14.14±2.18bc14.53±1.28bc13.29±2.17bc10.91±0.94cAlpha-amylase (U/mg prot)0.34±0.02a0.28±0.02a0.28±0.05a0.17±0.10b0.18±0.05b0.18±0.02b0.15±0.04bLipase (U/g prot)1.58±0.34c2.23±0.29bc3.40±0.47a2.04±0.13c3.13±0.94ab2.61±0.28abc2.25±0.15bc1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6.


## 3.6. Intestinal Morphology

The villus height (VH) of group FSBM4 was significantly higher than that of groups FSBM1 and FSBM2 (p<0.05). As to microvillus height (MH) and thickness of the intestinal wall (TIW), they showed decreasing order by groups FM, FSBM4, FSBM6, FSBM3, FSBM5, FSBM2, and FSBM1 (Table 12 and Figure 1), but no significant differences were observed in MH between groups FSBM4 and FM (p>0.05).Table 12
Middle intestinal histology parameters of juvenile turbot (Scophthalmus maximus L.) fed the experimental diets (means±S.E.M.)1.
Diets2FMFSBM1FSBM2FSBM3FSBM4FSBM5FSBM6VH (μm)3847.75±36.11ab607.72±23.48c645.94±24.76c714.26±37.80ab751.38±44.59ab874.48±76.68a818.37±47.61abTIW (μm)3158.38±3.78a92.43±2.37d101.89±10.63cd122.49±10.10bc128.67±4.19b115.88±7.26bc135.85±4.19bMH (μm)34.73±0.23a3.36±0.17c3.62±0.05bc3.87±0.06bc4.32±0.46ab3.82±0.29bc3.88±0.05bc1Data are means of triplicate. Means in the same row sharing the same superscript letter are not significantly different determined by Tukey’s test (p>0.05). 2Fish meal-based diet was named FM; diets with 450 g/kg of fish meal replaced by six kinds of commercial fermented soybean meal were, respectively, named FSBM1, FSBM2, FSBM3, FSBM4, FSBM5, and FSBM6. 3VH: villus height; TIW: thickness of the intestinal wall; MH: microvillus height.Figure 1
Representative intestine sections of juvenile turbot (Scophthalmus maximus L.) fed with (a) fish-meal-based diet FM and diets containing (b) FSBM1, (c) FSBM2, (d) FSBM3, (e) FSBM4, (f) FSBM5, and (g) FSBM6, all the sections were stained by haematoxylin and eosin. Scale bars, 100 μm.
(a)(b)(c)(d)(e)(f)(g)


## 4. Discussion

Numerous kinds of fermented soybean meal (FSBM) are now commercially available, whereas the nutritional profiles of these products vary with processing technologies, fermentation strains, and raw soybean meal, which would result in different effects on bioavailability of nutrients in aquaculture feeds [10, 11]. The present study showed that diets containing FSBM3 and FSBM4 yielded higher growth performance of juvenile turbot than diets containing other FSBM, suggesting their higher nutritional quality, whereas growth performance was significantly inhibited in fish fed diets with FSBM1 and FSBM2. According to previous studies on fish meal substitution, the reduced growth performance in replacement groups could generally be attributed to poor palatability, low digestibility, and imbalanced amino acid profile of alternative protein sources [28–30].The palatability of diets can be reflected by feed intake (FI) [31], and previous studies have revealed that the growth performance in fish was positively correlated with FI when dietary fish meal was substituted by FSBM [32, 33]. In the present study, no significant differences in FI were observed among dietary treatments FM, FSBM2, FSBM3, and FSBM5, which indicated that juvenile turbot had a better adaptability to these FSBM. Coupled with the trend in growth performance, it thus can be inferred that the FI was not responsible for growth reduction.The digestion and absorption, as important processes in dietary nutrient conversion, have important impacts on growth performance [34]. Apparent digestibility coefficient (ADC) provides an indirect measurement of dietary nutrient digestion [35]. As per Pearson’s correlation test, dietary protein utilization reflected by protein efficiency ratio (PER) and protein retention (PR) was positively correlated with the ADC of dry matter and protein. In the present study, the ADC for feed may be affected by digestive enzyme activity, antinutritional factors (ANFs), protein properties of FSBM, and intestinal structure. Results of this study showed that no significant differences in trypsin activity were observed among dietary treatments FSBM1, FSBM2, FSBM3, and FSBM4, while the alpha-amylase and lipase activities were higher in groups FSBM1 and FSBM2 than in FSBM3, which was opposite to the trend of feed digestibility. It thus could be deduced that the reduced digestibility in groups FSBM1 and FSBM2 was not attributed to the digestive enzyme activity, while the increased digestive enzyme activity in groups FSBM1 and FSBM2 might be a compensation for the decreased feed digestibility.Feed digestibility could also be affected by ANFs in soybean meal [36]. Trypsin inhibitor, as the main ANF in soybean meal, can resist the digestion of trypsin and stimulate the secretion of pancreatic juice, ultimately leading to endogenous amino acid depletion [37]. Apart from that, antigenic proteins, such as glycinin and β-conglycinin, are also important components of ANFs in soybean meal, which could lead to intestinal allergies and hinder the development of intestinal villi [38]. In this study, the trypsin inhibitor and glycinin contents of FSBM1 and FSBM2 were higher than those of the rest selected FSBM. Correspondingly, lower ADC of dry matter and protein was shown in diets FSBM1 and FSBM2. In contrast, higher feed digestibility was observed in the diet containing FSBM4 (with the lowest levels of glycinin and β-conglycinin). Within these detected ANFs, the trypsin inhibitor content in FSBM showed negative correlation with the PER, PR, and SGR, which indicated that the ANFs could inhibit growth and feed utilization by affecting feed digestibility. Thus, reducing the content of ANFs plays an important role in improving the quality of FSBM.According to previous studies, the protein properties of soybean meal can be greatly affected by fermentation treatment, where the high-molecular-weight protein could be degraded into low-molecular-weight protein and polypeptide [15]. Zheng et al. [39] reported that low-molecular-weight protein (<10,000 Dalton) reflected by water-soluble protein and trichloroacetic acid- (TCA-) soluble protein is more readily digested by aquatic animals. In the present study, the water-soluble protein and TCA-soluble protein contents of FSBM, which exhibited consistent trends with ADC of dry matter and protein, were both positively correlated with PER and PR. It could be deduced that the lower-molecular-weight protein in FSBM was responsible for the improved feed digestibility and protein utilization efficiency. Apart from that, the heating degree, reflected by the level of alkali- (KOH-) soluble protein, can also affect the protein properties of soybean meal [40]. Araba and Dale [27] revealed that alkali- (KOH-) soluble protein value in excess of 85% indicated underprocessing of soybean meal. For the underheated FSBM, it may contain more undenatured protein, which was not conducive to digestion. In the present study, the alkali- (KOH-) soluble protein content of FSBM1 (85.5%) and FSBM2 (86.5%) was significantly higher than that of the rest selected FSBM, indicating lower feed digestibility in groups FSBM1 and FSBM2 may also be attributed to high content of undenatured protein caused by underheated treatment. Therefore, improving the breakdown degree of FSBM protein and the content of low-molecular-weight protein is conducive to improve the utilization efficiency of FSBM in aquafeeds.The intestine is a major digestive organ for nutrients; thus, its structure has important effects on digestion [41, 42]. In the present study, the villus height (VH) was significantly lower in groups FSBM1 and FSBM2, which agree well with relatively lower feed digestibility and growth performance in both groups. Also, the microvillus height (MH) and thickness of the intestinal wall (TIW) showed the same pattern as ADC of protein, which was in agreement with some previous studies on Atlantic salmon, Salmo salar [43], Asian seabass, Lates calcarifer [44], and turbot [45]. It is thus deduced that different FSBM could result in varied effects on intestinal structure, thereby affecting the digestion capacity of turbot.Amino acid is the basic nutrient for protein metabolism, and an adequate supply of essential amino acid can facilitate protein anabolism and growth [34]. In the present study, each diet containing FSBM was supplied with crystalline L-lysine, L-methionine, and L-isoleucine to meet the essential amino acid requirements reported for turbot [46], which seemed that the dietary amino acids would not limit growth performance of turbot. Nevertheless, previous studies showed that 22%-80% of dietary crystalline amino acids could be leached in a few minutes after water immersion [47, 48]. Besides, the absorption of crystalline amino acids and protein-bound amino acids in the intestine is not synchronized [34, 47], which would compromise the efficiency of crystalline amino acids utilized by turbot. Thus, the original amino acid composition of FSBM may have crucial implications for growth performance. The isoleucine (Ile), phenylalanine (Phe), histidine (His), and arginine (Arg) levels of FSBM1 were all significantly lower than those of the rest selected FSBM, which was consistent with growth performance of fish fed diet with FSBM1. As to FSBM2, the content of its essential amino acids was not significantly different from that of FSBM3 and FSBM4, but the growth performance of fish fed diet with FSBM2 was significantly reduced, which may be attributed to the low digestibility of diet containing FSBM2. On the whole, the growth performance was significantly reduced in all the replacement groups as compared with the control, suggesting that the addition of crystal amino acids cannot make up for the lack of essential amino acids in FSBM, and further treatment (e.g., addition of coated amino acids and amino acids in peptide form) may be required to effectively improve the utilization efficiency of FSBM.In conclusion, this study demonstrated that commercially available FSBM3 and FSBM4 could yield higher growth performance of juvenile turbot than the rest selected FSBM when replacing 450 g/kg of fish meal in diets, while FSBM1 and FSBM2 significantly reduced the growth performance due to low digestibility and imbalanced amino acid supply. In aquafeed production, the content of water-soluble protein, TCA-soluble protein, and trypsin inhibitor could serve as relatively accurate indicators in the quality evaluation of FSBM.



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