Abstract

During the winter, the growth of amberjack, Seriola dumerili at aquaculture farms is significantly suppressed because of the decreased water temperatures. Serious economic losses are thus incurred at commercial aquaculture farms. To address this problem, we used microbial acid and alkaline proteases to enhance the digestibility of an extruder pellet (EP) diet to be used in the winter. We observed that the acid and alkaline protease activities at 15°C were significantly decreased compared to those at 20°C, but 77% and 44% activity of the acid and alkaline proteases, respectively, remained. We exposed the EP diet to glycine buffer (pH 3.0) with acid protease and/or Tris-HCl buffer (pH 8.0) with alkaline protease; the hardness of the EP diet was degraded by the addition of alkaline protease, and the weight decrease of the solid fraction of the EP diet was significantly changed to 10.5% as compared with control (7.4%). After reaction with a protease, the nitrogen content in the centrifugal supernatant was significantly increased by the addition of the enzymes, especially alkaline protease. With the oral administration of the EP diet containing alkaline protease to amberjack in the winter season, the fish growth was enhanced compared to those without protease.

Keywords:Amberjack; Protease; Low temperature; Extruded pellet

Introduction

Aquaculture is a global growth industry, but aquaculture feeds account for 60% of the total cost of an aquaculture farm’s management, and thus the reduction of the cost of feeds is urgently needed. Extrusion is one of the widely employed manufacturing process to prepare aqua-feeds and pet foods [1] because this process promotes hydrolysis of protein and starch, and improved growth rate of various aquaculture species [2-4]. In addition, the extrusion process promotes the release of soluble fiber by hydrolyzing cellulose and reduce the content of anti-nutritional factors [5-7]. Therefore, extruded pellet (EP) diet prepared through the process of extrusion is becoming a mainstream in aquaculture industry. On the other hand, there are some reports about non-effect or negative impact of EP on fish growth. In the case of bluefin tuna, it is known that the growth rate of fish fed with extruded pellet (EP) diets was lower than those fed with raw feeds [8]. In the case of diet which contains soybean meal for rainbow trout, the improvement of digestibility of carbohydrates by extrusion was not significantly observed because major carbohydrates are consists of oligosaccharides or non-starch polysaccharides [9]. Like this, there is room for improvement about application of EP diet to aquaculture.

Amberjack, Seriola dumerili is a marine pelagic species that is an important aquaculture species in the Japanese and commercial market because of its high growth rate and high commercial value [10,11]. South Kyushu and Shikoku Island, located in a warm region of Japan, are the best-known sites for the mass production of amberjack. Amberjack is distributed in tropical and subtropical marine areas [12], and warm temperatures are thus favorable for its growth. In recent years, the production of amberjack in Japan reached 40 thousand tons, and the production in South Kyushu, Kagoshima and Miyazaki accounts for >60% of the total production in Japan [13]. Amberjack lives also along Mediterranean coast, and recently Mediterranean countries focus on the aquaculture production of amberjack because of their adaptability to artificial conditions, high productivity and tastes based on market analysis [12,14-17].

Generally, the feeding activity of fish is affected by the water temperature [18]. The optimal temperature for the growth of amberjack is >26 °C, and the growth is mostly suppressed at <15 °C. The growth inhibition during the winter is a serious economic problem in amberjack aquaculture, and the enhancement of growth in this season is a logical way to decrease the total cost of amberjack production. In the case of yellowtail, Seriola quinqueradiata fed a formula diet such as EP diet during the winter, the growth and feed conversion ratio were lower than those observed in yellowtail fed raw fish-moist pellets due to the decreases in feeding frequency and protein digestibility caused by low temperatures [19]. Watanabe et al. [20] reported that growth performances of yellow tail were regulated by environmental water temperatures.

In Mediterranean countries also, the rearing trial of yellowtail or amberjack with feeding of semi-dry diets have been reported, but especially the information of EP diet to Mediterranean amberjack is scarce [10]. From now, it is predicted that the aquaculture production of amberjack with EP diet will be in high demand, and enhancement of growth rate based on improvement of digestibility of EP diet also will be essential for sustainable aquaculture fish production. In the past decade, the supplementation of exogenous enzymes in animal feeds including aquafeeds has attracted interests, and this approach improved feeds digestibility which resulted in enhancement of utilization of dietary nutrients [21-25]. However, the evidence about effect of supplementation of exogenous protease on the growth of amberjack is scarce, especially in winter season.

Toward the goal of enhancing the digestibility of feeds, proteases were first added to aquafeeds in the 1960s, and several proteases for aquaculture are now commercially available [26]. In the case of livestock and poultry, enhancements of growth and feed digestibility by supplementation with exogenous proteases were observed [27-31].

We conducted the present study to evaluate the effects of microbial proteolytic enzymes on (1) the decomposition of an EP diet at low temperature and (2) the growth of amberjack during the winter. We performed an in vitro test to confirm the decomposition of the EP diet by the addition of microbial proteases. We also conducted a rearing experiment with amberjack fed a diet containing microbial proteases, in order to evaluate the effect of protease on the growth of amberjack.

Materials and Methods

Effect of temperature on Protease activity

Commercial microbial proteases were used in this study. Bioplase 3LAP was obtained as the acid protease from Nagase ChemteX, Osaka, Japan. Amafeed P6 produced from Aspergillus melleus was obtained as the alkaline protease from Amano Enzyme, Aichi, Japan. The units of acid and protease activity were 25,000 IU and 50,000 IU per g of the product, respectively. Protease activity was determined by the measurement of the amount of liberated tyrosine from casein as a substrate.

As reaction buffers, we used 0.1 M glycine buffer (pH 3.0) and 0.1 M Tris-HCl buffer (pH 8.0) for the acid protease and alkaline protease, respectively. First, 100 μl of the crude enzyme solution was mixed with 500 μl of a 3.0% casein solution. The final enzyme activity was adjusted to 30 IU/100 μl in the reaction mixture. The mixture was incubated at 37°C for 10 min, and 500 μl of 5.0% trichloroacetic acid (w/v) was added to stop the enzyme activity. The mixture was centrifuged at 5,000 × g for 10 min, and the supernatant was used for the determination of tyrosine liberated from casein.

The supernatant (250 μl) was mixed with 0.55 mol/l sodium carbonate solution and incubated at room temperature for 10 min. Then, 125 μl of 660 mM Folin’s Phenol Reagent was added and incubated at 37°C for 30 min to generate measurable color based on the reaction with tyrosine. The absorbance of the supernatant at 660 nm was measured in a UV/VIS spectrophotometer (V-530, Jasco, Tokyo), and we calculated the amount of the liberated tyrosine by using a calibration curve previously made by a standard tyrosine solution. One unit of enzyme activity was defined as the amount of enzyme that would liberate the equivalent of 1 μg of tyrosine from casein per 1 min. The protein concentration of the crude enzyme solution was determined according to the method of Lowry et al. [32]. To evaluate the influence of temperature on the enzyme activity, we adjusted the reaction temperature to 15°C, 20°C, 25°C, 30°C and 35°C, and we compared the activities among the reaction temperatures.

The acid and alkaline proteases’ activity in a coexistent condition

The acid and alkaline proteases were mixed together in 0.1 M glycine buffer (pH 3.0) or 0.1 M Tris-HCl buffer (pH 8.0). In addition, the acid protease and the alkaline protease were each separately mixed in 0.1 M glycine buffer (pH 3.0) or 0.1 M Tris- HCl buffer (pH 8.0). The protease activity in each condition was determined by the same method as that described above.

Decomposition of the EP diet by addition of protease

An extruded Pellet (EP; diameter/ length 8 mm) diet named Hamachi Premium 8 (Chubushiryo Co., Ltd., Aichi, Japan) was used for this assay. The content of crude protein and crude fat were 49.3% and 10.2%, respectively. One pellet of the EP diet (approx. 0.5 g) was mixed in 0.1 M glycine buffer (pH 3.0) containing acid protease (final concentration, 160 IU/g of the EP diet) or 0.1 M Tris-HCl buffer (pH 8.0) containing alkaline protease (final concentration, 1000 IU/g of the EP diet). As a control, the mixture without proteases was prepared. The mixture was reacted at 15°C or 20°C for 3 h, and the mixture was centrifuged at 3,000 × g for 30 min. The centrifugal precipitate was dried at 105 °C for 6 h to remove the moisture, and we measured the weight of the dried precipitate to calculate the decomposition rate of the EP diet.

We mixed part of the centrifugal supernatant with the same volume of 20% trichloroacetic acid solution and reacted the mixture at 25 °C for 30 min to precipitate the protein in the solution. The reacted mixture was centrifuged at 5,000 × g for 30 min. The supernatant and the precipitate were collected as the TCA-soluble fraction and TCA-insoluble fraction, respectively. The nitrogen content of each sample was determined by the Kjeldahl method. The amino acid composition of the TCA-soluble fraction was analyzed with liquid chromatography-mass spectrometry (LC-MS) as described below.

To evaluate the coexisting acid and alkaline proteases on the decomposition of the EP diet, we first added both proteases to the EP diet under the 0.1 M glycine buffer and incubated the diet at 15°C for 3 h. We added an equal volume of 0.4 M Tris-HCl buffer to the mixture and incubated it at 15°C for 3 h. After the reaction, we analyzed each parameter, and the weight decrease of the solids and nitrogen content in each fraction by the same procedure as that described above.

Amino acid analysis

The amino acid composition of the TCA-soluble fraction was identified with a liquid chromatography-mass spectrometer (LCMS; UF-Aminostation, Shimadzu, Kyoto, Japan) consisting of a vacuum pump (Oil mist filter EMF20, Edwards, Crawley, UK), a gas generator (N2 model supplier 24F, System Instruments, Tokyo). The samples were mixed with an equal volume of 20% (w/v) trichloroacetic acid and centrifuged at 10,000 × g for 10 min. The mixture left for 30 min at room temperature. The supernatant was mixed with acetonitrile and used in an LC-MS analysis. As amino acid standard solutions, we used amino acid mixture standard solutions, type B and type AN-II (Wako Pure Chemicals, Osaka, Japan). L-glutamine, L-asparagine, and L-tryptophan solutions were separately prepared with reagents purchased from Nacalai Tesque (Kyoto, Japan). Derivatization of the amino acids was conducted with an APDSTag® Wako (Wako) at 60°C for 5 min.

Samples were injected into a shim-pack column (UF-AMINO, Shimazdu) (100 mm × 2.1 mm i.d., pore size 2 μm). The solvent system was APDSTAG Wako Eluent, acetonitrile for LC/MS, and APDSTAG Wako Borate Buffer (Wako), and the elution was carried out in gradient mode (total run 12 min), with a flow rate of 0.3 mL/min at ambient temperature.

Rearing experiment: amberjack feeding with protease-added EP diet

We obtained 100 amberjack S. dumerili with a mean weight of 550 g from a commercial fish farm (Miyazaki, Japan) and transported them to the Miyazaki Prefectural Fisheries Research Institute. The fish population was conditioned in 1,000 l - rearing tanks with vigorous aeration 2 weeks prior to the experiment. During the conditioning period, the fish were fed the control diet (Otohhime; Marubeni Nisshin Feed Co., Tokyo) twice daily at 1%– 2% of body weight.

Flow Three treatment groups were set: (i) the control group fed the control diet without proteases; (ii) the alkaline protease (AP) group fed a diet containing alkaline protease (1,000 IU/gdiet); and (iii) the acid and alkaline protease (AAP) group fed a diet containing both acid and alkaline protease (160 IU/g-diet and 1,000 IU/g-diet, respectively). Each group was raised in a separate raceway system. On 27 December 2014, we transferred 25 experimental fish into a separate raceway-1,000-ton tank, and they were reared in filtrated natural seawater with vigorous aeration. The fish were fed twice daily at restricted feeding ratio (1% – 2% of fish body weight) and reared for 90 days.

After 0, 30, 60, and 90 days of feeding, the body weight and body length of all fish was collectively measured. The collected fish were anesthetized by immersion of in 200 ppm phenoxyethanol solution with vigorous aeration. The body weight and the body length of each fish was quickly measured. At 90 days, the livers were dissected out from seven fish in each group and weighed for the calculation of the hepatosomatic index (HSI). The survival rate, the condition factor and the HSI were calculated as follows.
Survival rate (%) = (number of survived fish/initial number of fish) × 100
Condition factor = (body weight/body length3) × 100
HSI (%) = (liver weight/body weight) × 100

Statistical analyses

We analyzed the experimental data by performing a one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test (BellCurve for Excel, Social Survey Research Information, Tokyo). For the comparisons of pairs of groups, the data were subjected to Student’s t-test (Microsoft Excel 2013).

Results

Effects of temperature on the degradation of the EP diet

Figure 1 illustrates the protease activities at different reaction temperatures. The acid protease activity decreased as the temperature decreased. The alkaline protease activity was stable between 35°C and 20°C, and it was remarkably decreased at 15°C. Compared with the corresponding activities at 20°C, the activity at 15°C was decreased by 77% and 44% in the acid and the alkaline protease conditions, respectively.

The effects of the coexistence of acid and alkaline proteases on the enzyme activities

Figure 2 presents the protease activities in the solution containing a single protease or both proteases. Alkaline protease activity was detected only at pH 8.0 regardless of the supplementation a single protease or both proteases, and no significant difference between these two conditions was observed. Acid protease activity was detected only at pH 3.0 regardless of the supplementation with a single protease or both proteases, and no significant difference was observed between these two conditions.

The effects of the proteases on the decomposition of the EP diet

Table 1 presents the distribution of Kjeldahl-nitrogen in each fraction separated by centrifugation or TCA precipitation. In the case of the acid protease at 15° and 20°C, the weight of the solid content decreased and that of the nitrogen content increased in each fraction (except for the solid fraction) compared to those obtained when 0.1 M glycine-HCl buffer was used (control), but these results were not statistically significant. In the case of the alkaline protease at 20°C, the weight of the solid content decreased and that of the nitrogen content increased in each fraction (except for the solid fraction) compared to those obtained when 0.1 M Tris-HCl buffer was used (control).

In the case of the acid protease at 15°C, the weight of the solid content decreased and that of the nitrogen content increased in each fraction compared to those obtained with the use of 0.1 M glycine buffer (control), but these data were not significant. In the case of the alkaline protease at 15°C, the nitrogen content was increased in each fraction compared to those obtained with the 0.1 M Tris-HCl buffer (control). In the supernatant and TCA-insoluble fractions, significant differences of nitrogen concentration were observed compared to those obtained with the 0.1 M Tris-HCl buffer (control).

In the case of alkaline protease, the total amino acid content in the TCA-soluble fraction at 15°C was significantly higher than that obtained with 0.1 M Tris-HCl buffer (Figure 3). In the case of acid protease, no significance was observed (data not shown). Figure 4 shows the amino acid composition of the supernatant from the EP diet after incubation with the acid or alkaline proteases (TCA-soluble fraction). In the case of acid proteases, mainly glutamine was detected at >60% of the total amino acid content. As one of the essential amino acids for fish, threonine was detected at 12% of the total amino acid content. In the case of alkaline proteases, mainly histidine and taurine were detected at 26% and 14% in the total amino acid, respectively. As essential amino acids for fish, lysine, arginine, leucine, valine, phenylalanine was detected at >5%.

Table 2 summarizes the distribution of Kjeldahl-nitrogen in the samples reacted under the coexistence of both proteases. With the addition of proteases, the values in weight decrease of solids and nitrogen content in supernatant increased significantly compared to the control (i.e., absence of proteases). Visually, the texture of the EP diet was decomposed by the addition of proteases (Figure 5).

2q3Rearing trial of amberjack fed the EP diet with or without proteases

Figure 6 shows the changes in water temperature during the rearing trial. The water temperature was 18.3°C at the beginning of the rearing and gradually decreased with fluctuations and showed the lowest value (14.0°C) at 45 days. The temperature suddenly increased to 16.5°C at 60 days and ranged from 14.5°C to 17.0°C until 90 days.

The body weight of the amberjack increased with feeding in all groups (Figure 7A). At 60 days and 90 days of feeding, the average body weight in the AP group was highest, followed by the AAP group and then the control group. Regarding the body lengths of the fish, the AP group showed the highest average value at 90 days of feeding among the three groups (Figure 7B). In addition, the condition factor values were greatly decreased at 30 days of feeding in all groups. At 90 days of feeding, the average condition factor was highest in the AAP group, followed by the AP and control groups in that order (Figure 7C). Figure 8 illustrates the increase of total fish body weight per tank after 90 days of rearing. It was highest in the AP group, and lowest in the control group. In case of fish survival rate and HSI, there were no significant differences among groups (data not shown).

Discussion

In Miyazaki prefecture on Japan’s Kyushu Island, the water temperature seaside ranges from 16°C to 27°C yearly. In the present study, with the microbial acid protease used, the enzyme activity decreased gradually as the reaction temperature decreased (Figure 1). In the case of the alkaline proteases, the enzyme activity was stable at a wide range of temperature (20°C–35°C), and was greatly decreased at 15°C. Although the greatest reduction in enzyme activity was observed at 15°C (which is the lowest Miyazaki seaside temperature throughout the year), the activity remained at >40% against the maximum activity at higher temperatures. This result indicates that these enzymes are capable of working in aquafeeds during the winter season in Miyazaki.

In this study, the results of the in vitro test demonstrated that microbial acid and alkaline proteases enhanced the decomposition of the EP diet (Figure 3, Figure 4, Table 1 & Table 2). In the case of fungi, the highest activity of a protease from Aspergillus niger was observed at 35°C, and the activity decreased as the temperature decreased, as we observed with the acid protease in the present study [33]. In Chandrasekaran et al. study, the activity at 20°C decreased to < 20% from the maximum activity at 35°C. It was reported that the strongest activity of protease from mesophilic fungi was 35 °C [34,35].

The results of our rearing trial with amberjack indicated that the addition of proteases, especially alkaline protease, to the EP diet enhanced the growth of the fish during the winter season based on the improvement of the FCR. With our strategy, we expected a synergistic effect provided by the addition of both proteases. We speculate that with the oral feeding of the EP diet supplemented with both proteases, the diet first reaches the stomach of the fish, and then the acid protease works efficiently under a low pH condition. The diet then moves to the intestine by peristaltic motion, and the alkaline protease works efficiently in the intestine. However, we observed that the addition of acid protease to the diet with alkaline protease did not result in a growth-promoting effect.

In the in vitro test, the acid and alkaline proteases showed proteolytic activity at the adequate pH values 3.0 and 8.0, respectively in the coexistent condition (Figure 3). As one of the reasons that no effect occurred by the addition of acid protease, we note that the enzyme activity of acid protease per diet (160 IU) was lower than that of the alkaline protease (1000 IU). A second reason may be the different profiles of free amino acids produced by the decomposition of the diet. The in vitro test results revealed that the free amino acid composition after reaction with acid or alkaline protease was clearly different (Figure 5). In aquaculture fish species, 10 types of essential amino acids are known: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. These amino acids should be ingested via feeds because fish cannot synthesize them [36]. In our present use of acid protease, mostly glutamine was produced (Figure 5). In fish, glutamine is one of the non-essential amino acids because it can be synthesized by glutamine synthetase (GSase; EC6.3.1.2) [37]. We detected only threonine, isoleucine and phenylalanine with the use of the acid protease.

In contrast, in the case of alkaline protease, various essential amino acids including histidine, valine, leucine and phenylalanine were detected. Interestingly, mostly taurine was produced by the addition of alkaline protease. Taurine (2-aminomethanesulfonic acid) is known as a growth promoter in aquaculture species, and it is an abundant free amino acid in living organisms. The reported dietary requirement for taurine varied among marine finfish species and developmental stages, and the growth rate was improved by supplementation of taurine in the diet [38-40]. Taurine has various biological functions; not only in growth promotion but also in antioxidation, an immunostimulatory effect, osmotic regulation, and the prevention of green liver [41-43]. Sarih et al. [44] reported that taurine levels in diets of broodstock amberjack increase fecundity of eggs and maintain good fertilization rates. In live feeds stage of amberjacks, feeding of enriched rotifers with taurine significantly enhanced the growth of fish [45]. In the case of yellowtail juveniles, the weight gain and feed efficiency were improved by feeding with the taurine-supplemented diet, and additionally, taurine content in the muscle also increased depending on the dietary taurine level [46].

We also observed that arginine was produced from the diet containing alkaline protease. Arginine is a basic and abundant amino acid in fishery products [47]. In humans, arginine is considered a semi-essential amino acid, or a conditionally essential amino acid. There have been reports that arginine contributes to antioxidative mechanisms in various organisms [48], and arginine was reported to potentially regulate immune responses via the nitric oxide pathway [48-50]. Arginine plays an important role in endocrine functions such as adrenal and pituitary secretion [51].

Pohlenz et al. [52] reported that the combination of a vaccination and dietary supplementation with arginine and glutamate improved the immune function of channel catfish, and their resistance to the pathogenic bacterium Edwardsiella ictaluri was also enhanced. Consequently, Pohlenz et al. [53] reported that the growth and immune responses including phagocytosis in juvenile channel catfish (Ictalurus punctatus) were enhanced by dietary supplementation with arginine. In the case of yellowtail, Ruchimat et al. [54] reported weight gain in the fish, feed efficiency, protein efficiency ratio and nitrogen retention were improved with an increase in the level of dietary arginine up to 1.63%.

Conclusion

The results of our in vitro test showed that microbial alkaline proteases can enhance the decomposition of the EP diet and produce free amino acids. The results of our rearing experiment demonstrated that the oral administration of only alkaline proteases enhanced the growth of amberjack. We speculate that the reason for this is that the free amino acids produced by alkaline protease consisted mainly of various essential and functional amino acids including taurine and arginine.

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