Effects of undigested soybean protein on lipid digestion and growth of yellowtail fish, Seriola quinqueradiata

Abstract. An experiment was conducted to determine whether the undigested high molecular fraction (HMF) of soybean protein restricts the growth of yellowtail through impairment of lipid digestion and absorption. Soybean meal (SBM), soy protein isolate (SPI), digested SPI (DSPI) and digested and purified SPI (DPSPI) were used as experimental diets. Fingerling yellowtail fish were fed either a soybean protein or a fish meal (FM) diet for 8 weeks. The growth performance of DPSPI-fed fish was significantly better than that of fish fed other soybean proteins and was comparable to that of fish fed an FM diet. Plasma and tissue lipid levels of DPSPI-fed fish tended to be higher than in fish fed other soybean proteins. Significantly lower intestinal lipid levels and higher bile acid levels were found in DPSPI-fed fish when compared to fish fed SBM, SPI, and DSPI. These findings indicate that growth depression in yellowtail fish fed an SBM-based diet could be due to the negative effect the undigested HMF of soybean protein has on bile acid levels and lipid digestion in the fish.

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JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol. 58, No. 9, pp. 112-122 This paper is available online at EFFECTS OF UNDIGESTED SOYBEAN PROTEIN ON LIPID DIGESTION AND GROWTH OF YELLOWTAIL FISH, Seriola quinqueradiata Nguyen Phuc Hung1 and Peerapon Khaoian2 1Faculty of Biology, Hanoi National University of Education 2Faculty of Agriculture, Kochi University, Japan Abstract. An experiment was conducted to determine whether the undigested high molecular fraction (HMF) of soybean protein restricts the growth of yellowtail through impairment of lipid digestion and absorption. Soybean meal (SBM), soy protein isolate (SPI), digested SPI (DSPI) and digested and purified SPI (DPSPI) were used as experimental diets. Fingerling yellowtail fish were fed either a soybean protein or a fish meal (FM) diet for 8 weeks. The growth performance of DPSPI-fed fish was significantly better than that of fish fed other soybean proteins and was comparable to that of fish fed an FM diet. Plasma and tissue lipid levels of DPSPI-fed fish tended to be higher than in fish fed other soybean proteins. Significantly lower intestinal lipid levels and higher bile acid levels were found in DPSPI-fed fish when compared to fish fed SBM, SPI, and DSPI. These findings indicate that growth depression in yellowtail fish fed an SBM-based diet could be due to the negative effect the undigested HMF of soybean protein has on bile acid levels and lipid digestion in the fish. Keywords: Soybean protein, lipid digestion, bile acids, yellowtail. 1. Introduction Yellowtail fish, Seriola quinqueradiata, is one of the most produced and economically important aquaculture fish species in Japan. Much research has been conducted to evaluate the effect of replacing all or a part of the fish meal (FM) diet of yellowtail fish with defatted soybean meal (SBM). It has been found that an ‘excessive’ amount of SBM in a yellowtail diet resulted in inferior growth performance and physiological abnormalities [11]. Among the abnormalities were hypocholesterolemia, Received November 10, 2013. Accepted December 24, 2013. Contact Nguyen Phuc Hung, e-mail address: phuchungch10@yahoo.com 112 Effects of undigested soybean protein on lipid digestion and growth of yellowtail fish... which was reported to be a common symptom in this fish species which is fed SBM [6]. In red sea bream [4] and rainbow trout [9], a decrease in plasma cholesterol and triglyceride levels was observed in fish being fed SBM. Since the body lipid level is influenced by the efficiency of lipid digestion and absorption from the diet, these results suggest that fish receiving an SBM-based diet may have impaired dietary lipid digestion and absorption. To increase the feasibility of using soybean in yellowtail feed, it is essential to identify and remove those factors which are the cause of hypocholesterolemia in yellowtail. In mammals, it has been postulated that the undigested high molecular fraction (HMF) of soybean protein is the main cause of low plasma cholesterol levels [12]. However, this has never been investigated in fish. This present study aims to examine whether undigested HMF of soybean protein is responsible for restricting growth in yellowtail by impairing lipid digestion and absorption. 2. Content 2.1. Materials and methods * Soybean products Defatted soybean meal [SBM; crude protein (CP) 53%], soy protein isolate (SPI; CP 89%), digested SPI (DSPI; CP 90%) and digested and purified SPI (DPSPI; CP 95%) were used in this study. These soybean protein products were donated by Fuji Oil Co., Ltd. (Osaka, Japan). DSPI and DPSPI were prepared as follows: SPI was added to water (10:1, crude protein:water) followed by artificial digestion with commercial protease (AY10; Amano Enzyme, Aichi, Japan) derived from Bacillus species. The protease was added to reach 4% SPI by weight and hydrolysis was carried out at 50 ◦C. After 5 h, this digested material was heated at 85 ◦C for 30 min to deactivate and sterilized the enzyme. The digested material was then cooled to 15 ◦C and spray-dried to produce peptides containing undigested HMF and digested low molecular peptides (DSPI). The product of protease decomposition was centrifuged (1,500 rpm for 30 min) to remove the HMF, and consequently a supernatant was filtrated (0.45 µm; Advantec Tokyo, Tokyo) and then freeze-dried to produce peptides containing only the digested low molecular peptides (DPSPI). * Diet preparation Five isonitrogenous and isoenergetic experimental diets were formulated with FM and the four soybean products (Table 1). Crystalline methionine (0.5 g/100 g diet) was added to the soybean protein-containing diets to meet the methionine requirement of fingerling yellowtail [10]. After the powdered ingredients were thoroughly mixed with pollock liver oil, water was added to produce a stiff dough. The dough was then pelletized using a laboratory pellet mill and stored at -30 ◦C until used. 113 Nguyen Phuc Hung and Peerapon Khaoian * Fish and rearing conditions The experiment was carried out at Usa Marine Biological Institute, Kochi University (Kochi, Japan). A total of 250 fingerling yellowtail (mean body weight 20.3 g) were randomly distributed in 10 indoor FRP tanks (800 l holding capacity, 2 tanks/diet, 25 fish/tank). Fish were hand-fed the experimental diets to apparent satiation twice a day (8:30 a.m. and 4:00 p.m.), 6 days a week for 8 weeks. Dissolved oxygen and water temperature were monitored daily and ranged from 4.8 ppm to 6.9 ppm and 23.3 ◦C to 28.1 ◦C, respectively. * Sampling At the end of the feeding trial, fish were starved for 24 h before sampling. Fish were anesthetized with 400 ppm 2-phenoxyethanol and weighed individually. Blood samples from 6 fish in each tank were taken from the ventral aorta using 1-ml heparinized syringes. Plasma was separated by centrifugation at 10,000 rpm for 10 min. These 6 fish were then dissected to collect gallbladder, liver, and muscle samples. The remaining fish were returned to their original tanks where they continued to eat the experimental diets. After 1 week, digesta from the anterior and posterior intestines were taken from 6 fish in each tank at 4 h after feeding. The intestinal tract was divided as described by Murashita [8]. All the samples were stored at -30 ◦C until analyzed. * Analytical methods Plasma was analyzed for total cholesterol, triglycerides, nonesterified fatty acids (NEFA) and phospholipids using a commercial human diagnostic assay kit (Wako Pure Chemical Industries, Japan). These lipid components were also analyzed in freeze-dried intestinal digesta. The lipids were extracted from 20 mg of digesta with 5 ml chloroform:methanol (2:1, v/v) according to the method described by Folch [3]. The solvent and digesta mixture was allowed to stand for 6 h at room temperature, then centrifuged at 3000 rpm for 10 min to separate the supernatant from the residue. The solvent was evaporated under vacuum with a rotary evaporator (Tokyo Rikakikai Co., Ltd.) and the lipids were dissolved in 500 µL Triton X-100 (5 g/100 mL). The dissolved lipid solution was assayed according to the diagnostic kit procedures (Wako Pure Chemical Industries, Japan) for quantification of lipid components. Total bile acid was extracted from freeze-dried digesta as described by Beher [1]. Approximately 15 mg digesta were added to 1 mL aqueous ethanol 90%, then left for 2 h at room temperature. After centrifugation at 3000 rpm for 10 min, 200 µL of the supernatant was used for total bile acid analysis using the diagnostic kit procedures (Wako Pure Chemical Industries, Japan). Extract from bile juice was further diluted 1200-fold with distilled water and total bile acid level was quantified using the same diagnostic kit as mentioned above. Total lipid levels in liver and muscle were determined gravimetrically after extraction with chloroform:methanol (2:1, v/v) according to the method described by Folch [3]. The taurine content in liver, muscle, and the experimental diets were extracted using 114 Effects of undigested soybean protein on lipid digestion and growth of yellowtail fish... 5% trichloroacetic acid [7], then quantified by high performance liquid chromatography (model L-2000, Hitachi). An approximate composition of the experimental diets was determined according to the AOAC method [16]. Table 1. Formulation and approximate composition of the experimental diets Ingredients (%) FM SBM SPI DSPI DPSPI Soybean meal 0 49.5 0 0 0 Soy protein isolate 0 0 30 0 0 Digested SPI 0 0 0 30 0 Digested and purified SPI 0 0 0 0 28 Fish meal 71 33 33 33 33 Fish oil 10 10 10 10 10 Cellulose 12 0 9.5 9.5 11.5 Starch 0 0 10 10 10 Methionine 0 0.5 0.5 0.5 0.5 Vitamin mixture (1) 2 2 2 2 2 Mineral mixture (2) 2 2 2 2 2 Guar gum 0.5 0.5 0.5 0.5 0.5 CMC-Na (3) 2.5 2.5 2.5 2.5 2.5 Proximate composition (dry matter basis) Crude protein (%) 51.4 50.6 49.6 50.1 50.5 Crude lipid (%) 16.3 14.8 14.5 14.1 14.3 Ash (%) 12.5 10.3 8.4 7.9 8.1 Taurine (g/kg) 2.5 1.4 1.2 1.3 1.3 (1) Vitamin mixture containing (mg/100 g dry diet): thiamine HCl, 2.4; riboflavin, 4.4; pyridoxine HCl, 2.4; folic acid, 2.4; nicotinic acid, 7.2; calcium pantothenate, 14; biotin, 7.0; inositol, 169; choline chloride, 1168; calcium ascorbate, 178; cyanocobalamin,1.6; vitamin A palmitate, 4.0; vitamin D3, 0.0045; vitamin E (DL-α-tocopherol), 176; menadione–NaHSO4 , 5.13. (2) Mineral mixture containing (mg/100 g dry diet): KH2PO4, 412; Ca(H2PO4)2.H2O, 618; calcium lactate, 282; iron proteinate, 166; ZnSO4.H2O, 9.99; MnSO4.H2O, 6.3; CuSO4.H2O, 2.0; CoSO4.7H2O, 0.05; KIO3, 0.15. (3) CMC-Na: sodium carboxymethyl cellulose. * Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA). Statistical differences between groups were assessed using the Tukey-Kramer test and significance was based on a 5% level of probability. 115 Nguyen Phuc Hung and Peerapon Khaoian 2.2. Results 2.2.1. Growth performance Growth and feed performance of fingerling yellowtail fed the experimental diets are shown in Table 2. Final body weight tended to be lower in fish fed SBM, SPI and DSPI than in fish fed FM, and the difference between SPI- and FM-fed fish was significant. However, final body weight of fish fed DPSPI was similar to that of fish fed FM. A similar trend was found for the specific growth rate (SGR) of fish. Significantly inferior feed conversion ratios (FCR) were observed in fish fed SBM, SPI and DSPI compared with those fed FM and DPSPI (P < 0.05), while FCR values were comparable in the latter 2 groups. Feed intake of fish fed SBM, SPI and DSPI was significantly higher than that of fish fed FM while no statistical difference was found between fish fed DPSPI and FM. Table 2. Growth and feed performance of yellowtail fed the experimental diets (1) Dietary groups Parameters (2) FM SBM SPI DSPI DPSPI Initial BW 20.3±1.9 20.2±1.9 20.2±2.0 20.3±2.2 20.3±1.8 Final BW 117.1±12.5b 106.8±11.7ab 104.3±9.4a 107.5±10.7ab 116.1± 11.9b SGR 6.26±0.03c 5.94±0.02ab 5.86±0.02a 5.95±0.02ab 6.21±0.30bc FCR 1.10±0.03a 1.23±0.01b 1.41±0.01c 1.23±0.01b 1.16±0.05a Feed intake 5.53±0.15a 5.99±0.16b 6.53±0.04c 5.99±0.05b 5.80±0.06ab (1) Values are mean ± standard deviation (n = 2). Values of each parameter in the same row with different superscripts are significantly different (P < 0.05). (2) Body weight (BW) is calculated in g; SGR as %BW day−1; feed intake as %BW.day−1. SGR = 100 × (ln final wt – ln initial wt) × duration in days−1. FCR = total dry feed intake × wet weight gain−1. Feed intake = 100 × total dry feed intake × [(initial BW + final BW)/2]−1 × days fed−1. 2.2.2. Plasma lipid levels Table 3. Plasma lipid levels at 24 h after feeding yellowtail the experimental diets (1) Dietary groups Lipids (2) FM SBM SPI DSPI DPSPI Total cholesterol 329.2±32.2b 250.0±31.1a 257.0±30.6a 252.7±32.5a 254.5±24.1a Triglyceride 114.1±16.3 109.8±18.2 111.3±14.2 111.0±14.8 110.1±14.9 NEFA 0.52±0.07b 0.39±0.06a 0.44±0.06ab 0.38±0.08a 0.48±0.09b Phospholipid 727.0±96.3b 548.0±61.9a 565.5±63.8a 543.0±62.4a 600.5±63ab 116 Effects of undigested soybean protein on lipid digestion and growth of yellowtail fish... (1) Values are mean ± standard deviation (n = 12). Values of each parameter in the same row with different superscripts are significantly different (P < 0.05). (2) Total cholesterol, triglyceride and phospholipid are calculated as mg/dL; NEFA as mEq/L. Plasma lipid levels of yellowtail at 24 h after feeding are shown in Table 3. Total cholesterol levels were similar in fish fed all 4 soybean proteins and were significantly lower than those fed FM (P < 0.05). There were no significant differences in triglyceride level among the treatments, whereas NEFA and phospholipid levels of fish fed DPSPI and FM were higher than those of fish fed SBM, SPI and DSPI. 2.2.3. Lipid contents in posterior intestinal digesta Lipid contents in intestinal digesta of fish fed DPSPI were significantly lower than those of fish fed other soybean proteins (Table 4). However, there were no significant differences in intestinal lipid contents between fish fed DPSPI and FM, except total cholesterol. Table 4. Lipid contents in posterior intestinal digesta at 4 h after feeding yellowtail the experimental diets (1) Dietary groups Lipids (2) FM SBM SPI DSPI DPSPI Total cholesterol 1.1 ± 0.1b 1.6 ± 0.4c 1.6 ± 0.1c 2.0 ± 0.4c 0.9 ± 0.1a Triglycerides 7.5 ± 1.9a 29.0 ± 4.1bc 25.1 ± 4.9b 38.4 ± 9.5c 5.1 ± 0.6a NEFA 5.2 ± 0.5a 11.1 ± 1.2b 10.3 ± 0.3b 12.5 ± 1.9b 3.9 ± 0.6a Phospholipids 2.6 ± 0.4ab 9.2 ± 3.9c 5.6 ± 0.8bc 6.4 ± 1.3bc 1.2 ± 0.3a (1) Values are mean ± standard deviation (n = 12). Values of each parameter in the same row with different superscripts are significantly different (P < 0.05). (2) Total cholesterol, triglyceride and phospholipid are calculated as mg/g dry matter; NEFA as mEq/100g dry matter. 2.2.4. Total bile acid contents in intestinal digesta and gallbladder Total bile acid contents in the anterior and posterior intestines of fish are shown in Figure 1. Fish fed DPSPI showed significantly higher total bile acid values (P < 0.05) in the anterior intestine compared with fish fed other soybean proteins. On the other hand, total bile acid values of fish fed the 4 different soybean proteins were lower than that of fish that were fed FM. As shown in Figure 2, significantly lower total bile acid contents in the gallbladder were found in fish fed SBM, SPI and DSPI compared with those fed FM, whereas the content of DPSPI-fed fish was comparable to that of FM-fed fish. 117 Nguyen Phuc Hung and Peerapon Khaoian Figure 1. Total bile acid contents in anterior and posterior intestinal digesta at 4 h after feeding yellowtail the experimental diets Values are mean and standard deviation (n = 12). Different letters within each intestinal part denote significant differences (P < 0.05). Figure 2. Total bile acid contents in gallbladder at 24 h after feeding yellowtail the experimental diets Values are mean and standard deviation (n = 12). Different letters within each intestinal part denote significant differences (P < 0.05). 2.2.5. Lipid contents in liver and muscle Crude lipid contents of fish fed DPSPI were higher than those of fish fed SBM, SPI and DSPI in both liver and muscle (Table 5). Although the crude lipid content in liver of 118 Effects of undigested soybean protein on lipid digestion and growth of yellowtail fish... fish fed DPSPI was lower than that in fish fed FM, no such difference was found in the crude lipid content of muscle between these 2 groups. Table 5. Crude lipid contents in liver and muscle of yellowtail fed the experimental diets (1) Dietary groups Lipids (2) FM SBM SPI DSPI DPSPI Liver 102.2 ± 4.7c 67.9 ± 3.2a 69.7 ± 2.8a 66.9 ± 2.8a 80.8 ± 3.7b Muscle 25.9 ± 2.6b 19.7 ± 1.6a 20.4 ± 1.5a 20.3 ± 2.6a 23.7 ± 1.6b (1) Values are mean ± standard deviation (n = 12). Values of each parameter in the same row with different superscripts are significantly different (P < 0.05). (2) Crude lipid is calculated as mg/g wet tissue. 2.2.6. Taurine contents in liver and muscle As shown in Figure 3, taurine levels in both liver and muscle of fish fed all soybean proteins were lower than those of fish fed FM. Taurine levels in fish fed DPSPI tended to be higher than those in fish fed the other soybean proteins. However, no significant differences in taurine levels were detected between SPI- and DPSI-fed fish in liver, or between SBM- and SPI-fed fish in muscle. Figure 3. Taurine contents in liver and muscle of yellowtail fed the experimental diets Values are mean and standard deviation (n = 12). Different letters within each intestinal part denote significant differences (P < 0.05). 2.3. Discussion In the present study, growth performance in fish fed DPSPI was better than in fish fed other soybean proteins (SBM, SPI and DSPI) and was comparable to that of 119 Nguyen Phuc Hung and Peerapon Khaoian fish fed FM (Table 2). Low feed intake has been reported to be one reason for the low growth performance in fish fed plant protein-based diets [2]. In the present study, the feed intake of yellowtail fed SBM, SPI and DSPI was higher than that of fish fed DPSPI and FM. Therefore, low growth performance observed in fish fed SBM, SPI and DSPI was not due to a feed intake decrease. It has been reported that FM is rich in taurine plant protein sources including soybean are a poor source of taurine [15], and taurine supplementation markedly improved growth performance of yellowtail being fed soy protein concentrate-based diets [13]. In the present study, however, tissue taurine content did not differ among fish fed SBM, SPI, DSPI and DPSPI, and was lower than in fish fed with FM (Figure 3). This suggests that the low taurine content of soybean products is not the only factor reducing growth performance of yellowtail fed soybean protein. Since DPSPI differs from other soybean proteins in the amount of undigested HMF, the presence of undigestedHMF in fish fed other SBMs could be responsible for impaired growth rates. In the present study, yellowtail fed SBM, SPI and DSPI showed significantly higher lipid contents in posterior intestinal digesta than did fish fed DPSPI (Table 4). On the other hand, the growth and lipid contents in muscle and liver of fish fed SBM, SPI and DSPI were lower than those in fish fed DPSPI and FM (Table 5). Since body lipid level is influenced by the efficiency of lipid digestion and absorption from the diet, the results of these studies suggest that fish fed SBM, SPI and DSPI have poor dietary lipid digestion and absorption, and the poor dietary lipid digestion and absorption is a factor responsible for the low growth observed in fish fed with these soybean proteins. In the present study, total bile acid levels in the gallbladder and anterior intestine of fish fed SBM, SPI and DSPI were lower than those of fish fed DPSPI (Figures 1 and 2). Bile acid is synthesized from cholesterol and is conjugated with taurine or glycine before being stored in the gallbladder [14]. In yellowtail, conjugation is exclusive to taurine, and cholyltaurine and chenodeoxycholyltaurine are the main bile acids [5]. Thus, lower bile acid levels found in the gallbladder and anterior intestine of SBM-, SPI-, and DSPI-fed fish could be the result of low body taurine levels. On the other hand, bile acid levels in gallbladders of DPSPI-fed fish were comparable to those of FM-fed fish even though taurine in their body tissues was not higher than in fish fed other soybean proteins. This could be due to a better bile acid reabsorption rate in DPSPI-fed fish compared with fish fed other soybean proteins. The bett
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