Effects of diets microencapsulated with different wall materials on growth and digestive enzymes of the larvae of Penaeus japonicus bate.
KEY WORDS: digestive enzyme, gelatin, ethyl cellulose, microencapsulated diet, larvae, Penaeusjaponicus
The expense, unpredictable availability, and unsatisfactory nutrient delivery from live food highlight the importance of microdiets to replace live food completely or in part. The diet for larvae must meet the nutritional requirement of fast growth under the condition of an incomplete functional digestive system during ontogeny. The low digestive capacity of larvae and the absence of a functional stomach before metamorphosis have been pointed out as factors responsible for a poor performance of microdiets. Many authors have hypothesized that poorly developed digestive enzymes and low digestive enzyme activity explain the failure of the microdiet (Cahu & Zambonino Infante 2001). In some species, enzymes involved in the digestion of proteins, lipids, and carbohydrates were present in larvae before exogenous feeding commenced (Lazo et al. 2007). Low levels of trypsin, amylase, and alkaline phosphatase activity were detected during the larval stage, and this enzymatic activity increases with age (Gisbert et al. 2009). An understanding of enzyme availability is especially important when considering microdiet development (Johnston et al. 2004). Although larvae are able to ingest a microdiet, limited success has been obtained when the microdiet was used as the sole food source and the digestive enzyme activity of the larvae was similar to that of malnourished or starving larvae. Marine fish larvae can use compound diets as early as mouth opening if the digestive specificity of larvae is taken into account in the diet composition at early stages of development (Cahu & Zambonino Infante 2001). Recent studies have evaluated the effect of diet on digestive enzyme activity in larvae to develop successful microdiets designed to replace live food (Kolkovski 2001, Kotzamanis et al. 2007, Zambonino Infante & Cahu 2007).
Nutrient leaching is one of the main problems encountered when developing a microdiet for fish larvae. Water-soluble micronutrients, which are indispensible for the survival and growth of the larvae, are easily lost when immersed in water. A lot of formulated diets have been tested, but limited success is reported (see the review by Langdon (2003)). A microbound diet is widely used, and binder composition and amount are important considerations in the development of a microdiet. Binder type could affect the attractiveness, as well as palatability and digestibility, of the microdiet (Partridge & Southgate 1999, Guthrie et al. 2000, Genodepa et al. 2007). Diets containing too much binder influence digestibility; however, diets containing too little binder influence the stability in water, resulting in deterioration of water quality and loss of valuable dietary nutrients. A microencapsulated diet has proved to be an efficient way to retain water-soluble nutrients. However, scant information was reported about the type and amount of wall material influencing the digestibility of the diet and digestive enzyme activity of the larvae. Originally developed as a pharmaceutical technique, the fluidized bed coating is now increasingly being applied in the food industry to fine-tune the effect of functional ingredients and additives (Dewettinck & Huyghebaert 1999).
In the current study, we produced diets microencapsulated with gelatin and ethyl cellulose using the fluidized bed coating process. The characteristics of the diets microencapsulated with different wall materials were compared, and growth performance and activity of the digestive enzymes of Penaeus japonicus larvae fed the microencapsulated diets were tested. The purpose of this study was to formulate a more balanced and highly efficient feed for the larvae.
MATERIALS AND METHODS
Raw materials for feed formulation included fish meal, casein, peanut meal, wheat flour, soy lecithin, fodder yeast, papain, fish oil, vitamin premix, mineral premix, and so on (Xie et al. 2010). All the raw materials were supermicromilled, then were mixed thoroughly. The mixture was used as the core material, and gelatin and ethyl cellulose were used as wall materials for the coating process. The microcapsules were prepared using a fluidized bed coating process (Xie et al. 2010). A total of 200 g gelatin in water and 100 g ethyl cellulose in alcohol was dissolved and used for coating the suspension, respectively. The proximate analysis of the microencapsulated diet was determined according to AOAC (1990) methods and is presented in Table 1.
Characteristics of the Microencapsulated Diet
The particles were separated into different classes by sieving. Bulk density is the weight of an ingredient per unit volume. Bulk density was determined by filling a 1-qt. container and weighing the amount of samples to fill the container. The microencapsulated diets were also submitted to scanning electron microscopy (QVANTA-200; FEI Inc., Eindhoven, Holland) to determine coating integrity. Inclusion efficiency, lipid encapsulation efficiency, and nitrogen retention efficiency were measured according to Xie et al. (2010). The pH value of the microencapsulated diets was determined as follows: Triplicate samples of the diet (10 g) were dissolved in 100 mL solution. A pH meter (model SP-71; METTLER TOLEDO, Inc., Shanghai, China) was used to determine pH values. The solutions were distilled water, 35[per thousand] NaCl, and seawater with a pH of 8.2, respectively.
Larval Rearing and Experiment Design
The larval rearing experiment is illustrated in Xie et al. (2010). There were 4 treatments and each treatment had 3 replicates:
1. Control: 50% shrimp flake + 50% Artemia
2. Group I: 50% diet microencapsulated with gelatin + 25% shrimp flake +25% Artemia
3. Group II: 100% diet microencapsulated with gelatin
4. Group III: 100% diet microencapsulated with ethyl cellulose
Growth Performance and Body Composition
Ten larvae per tank were randomly sampled 30 days after hatching to obtain final weight and total length. At the end of the experiment, survival was determined by counting the individuals in each tank. Samples for body composition were freeze-dried. Proximate composition of the larvae was determined according to AOAC (1990) methods.
Analytical Methods for Digestive Enzymes
After collection, larvae were starved for 24 h to allow any remaining food in the gut to be assimilated, to minimize the potential effects of exogenous enzymes from undigested feed in the gut. The specimens were thoroughly rinsed in distilled water, then immediately frozen and stored at -80[degrees]C until enzyme activity assay. The whole larva was homogenized in appropriate ice-cold, deionized water for enzymatic assay. Suspensions were centrifuged by refrigerated centrifuge (3K30; Sigma, Germany) at 8,000g for 20 min at 4[degrees]C, then incubated with substrate and read on a spectrophotometer (UV2100; Unico Instruments Co., Ltd., Shanghai, China). All measurements were carried out in triplicate and finished within 24 h.
Trypsin activity was measured according to Pan et al. (2005). suspensions were incubated with 1 mL 2% casein solution and 5 mL 0.02 M phosphate buffer (0.05 M Tris, pH 7.5) at 37[degrees]C for 10 min. Then 1 mL enzyme solution was added and incubated in a bath for 10 min at 37[degrees]C. After terminating the reaction with 2 mL 0.4 M trichloroacetic acid, all the reagents were centrifuged at 4,000g for 15 min at 4[degrees]C. A total of 1 mL filtrate was then incubated with 5 mL 0.4 M sodium carbonate and 1 mL Folin reagent at 37[degrees]C for 10 min. The activity unit (units per milligram of protein) was defined as 1 [micro]g tyrosine released by hydrolyzed casein/rain at 660 nm.
Amylase activity was measured according to Uys and Hecht (1987). Samples of the supernatant were incubated with 1% soluble starch as substrate in the same phosphate buffer (pH 7.5). Production of reducing sugars was qualified colorimetrically after adding the dinitrosalicylic acid reagent (1% 3,5-dinitrosalicylic acid). A standard curve was prepared using maltose solution instead of the enzyme sample. One unit (unite per milligram protein) was defined as 1 mg maltose equivalents liberated per min at 37[degrees]C.
Alkaline phosphatase activity was assayed according to Bessey et al. (1946) using p-nitrophenyl phosphate as a substrate in 30 mM Na2CO3 buffer (pH 9.8, 10 min, 37[degrees]C). The reaction was stopped with 3 M NaOH. One unit (unit per milligram protein) was defined as 1 [micro]g p-nitrophenyl released per min at 405 nm.
The protein concentration of the enzyme extracts was determined by the Bradford (1976) method using bovine serum albumin as a standard protein.
Results are presented as mean [+ or -] SD. All statistical analyses were carried out using the SPSS statistical package (SPSS Inc., Chicago, IL). Tank and treatment effects were evaluated by 1-way analysis of variance. Differences were considered statistically significant if P < 0.05.
Scanning Electron Microscopy
Figure 1 showed the surface was a uniform and continuous film around the core. The core material was irregular in shape; therefore, the film thickness was not uniform and it varied along the surface. The surface of the diet microencapsulated with ethyl cellulose was somewhat white, because ethyl cellulose is a white powder.
[FIGURE 1 OMITTED]
Feed Particle Size and Bulk Density
There was no significant agglomeration in the coating process, and the diameters of the two different microencapsulated diets were in a normal distribution and within a broad size range (Table 2). The size of the diet microencapsulated with gelatin with 50% was between 250 [micro]m and 590 [micro]m, but the size of the diet microencapsulated with ethyl cellulose with 50% was between 178 [micro]m and 420 [micro]m. The bulk density of the diet microencapsulated with gelatin was greater than that of the diet microencapsulated with ethyl cellulose.
Inclusion, Lipid Encapsulation, and Nitrogen Retention Efficiency
The inclusion efficiency, lipid encapsulation efficiency, and nitrogen retention efficiency of the diets microencapsulated with different wall materials are presented in Table 3. From the results, despite the less amount of wall material used, the diet microencapsulated with ethyl cellulose had better performance with regard to lipid encapsulation efficiency and nitrogen retention efficiency compared with the diet microencapsulated with gelatin. This can attribute to the fact that the wall material comprised of ethyl cellulose could not dissolve in water. The results suggest that wall material had a significant influence on lipid encapsulation efficiency and nitrogen retention efficiency of the microencapsulated diets.
The pH values of the two different microencapsulated diets in different solutions are presented in Table 4. The pH value of the microencapsulated diet was slightly acidic; wall material had no significant effect on the pH value of the microencapsulated diet. The difference was because nutrients can be released more easily from the diet microencapsulated with gelatin compared with the diet microencapsulated with ethyl cellulose.
Growth Performance and Body Composition
The growth performance and body composition of the larvae are presented in Table 5. Growth and survival data showed that the experimental diets were all ingested, digested, and assimilated by the larvae. The microencapsulated diets prepared in this study showed a low leaching and sinking rate. The wet weight of larvae increased 313.4% when fed the diet microencapsulated with ethyl cellulose from 10 up to 30 days after hatching.
The survival, total length, and wet weight of larvae were greater in the microencapsulated diet groups (group II and group III) compared with the control (P < 0.05). There was no significant difference in survival and wet weight of the larvae between the gelatin-wall microencapsulated group (group II) and the ethyl cellulose-wall microencapsulated group (group III; P > 0.05). However, there was a significant difference in total length of the larvae between the two groups.
Digestive Enzyme Activity
The main enzyme activity of the larvae is presented in Table 6. The trypsin and amylase activity of larvae in groups II and III was higher than that of the control (P < 0.05). There was no significant differences in amylase activity between group II and group III (P > 0.05), but there was a significant difference in trypsin activity between group II and group III (P < 0.05).
The highest trypsin activity of the larvae was found in group III, which was fed the diet microencapsulated with ethyl cellulose; the lowest trypsin activity was found in the control group. The highest amylase activity of the larvae was found in group l and the lowest amylase activity was found in the control. There was no significant difference in alkaline phosphatase enzyme activity of the larvae between each group (P > 0.05).
Gelatin can be used as a protein source and a binder in microbound diets (Kolkovski & Tandler 2000). Ethyl cellulous is widely used as a coating material for powder controlling release (Watano et al. 2004). Ethyl cellulose is not included in the feed for aquatic larvae generally and the diet microencapsulated with ethyl cellulose was feasible for larval shrimp in this study. During the fluidized bed coating process, the gelatin water suspension increased the moisture and crude protein content of the microdiet; however, the ethyl cellulose-alcohol coating suspension decreased the moisture and crude protein content of the microdiet. When immersed in water, the microdiet suffered from dissolution, swelling, cracking, and penetration. As a result of the high surface-to-volume ratio, high efficiency to retain the water-soluble nutrients becomes rather difficult. This needs further improvement of the manufacturing technique of the microdiet. Gelatin can be inflated when immersed in water to keep its buoyancy, so the diet microencapsulated with gelatin had a soft, deformable wall in the water column. Compared with the diet microencapsulated with gelatin, the diet microencapsulated with ethyl cellulose had less inflation and sank faster in the water column.
Unlike microencapsulated diets, microbound diets lack a true capsule wall, which may aid in microbound diet digestion. For core material in the microcapsule to be assimilated by fish larvae, the core must be released either by enzymatic digestion or physical digestive processes from the microcapsule. Lipid-wall and protein-wall microencapsulated diets for larvae have been reported (Langdon 2003), but the payload is rather low and too much lipid hampered absorption. Considering the characteristics of 2 wall materials and the balance of feed formulation, 7.8% gelatin and 4.2% ethyl cellulose to the whole microencapsulated diet were adopted as coating material, respectively; the payload was high and the amount of wall material was low. In a microbound diet, a binder must be easily digested by fish larvae and also chemically attractive in itself or capable of leaching feed attractant. Many fish larvae cannot digest hard, thick-wall capsules (Fernandez-Diaz & Yufera 1995). Moreover, microdiets contain 60-90% dry matter compared with only 10% in zooplankton. This may lead to insufficient digestibility, because it is difficult for larvae to break down dry hard particles than live food. As for microencapsulated diets, different wall materials and the amount of wall material should be considered on the ingestion, digestion, and assimilation of the microdiet. This study suggests that the amount of 7.8% gelatin and 4.2% ethyl cellulose in the microencapsulated diet had no significant influence on the growth and survival of the larvae. The reason for the high growth rate in group III larvae could be attributed to the fact that the ethyl cellulose-wall microencapsulated diet has a higher retention efficiency compared with the gelatin-wall microencapsulated diet.
It has been assumed that the gut pH in larvae is determined by the surrounding seawater and therefore is slightly alkaline. Little is known about the capability of fish larvae to regulate gut pH. The pH value of the microdiet can influence the water stability of the microdiet. There is little knowledge of the optimum pH value of the microdiet for larvae. Different diets influenced the gut pH of the larval turbot (Hoehne-Reitan et al. 2001). To our knowledge, the acid diet contributed to the digestion (Yufera et al. 2004). In this study, the pH value of the microencapsulated diet was slightly acidic and good for digestion.
Early research relied on growth and survival data to assess the worth of an experimental diet. Recently understanding digestive enzyme availability was especially important when considering microdiet development. Many kinds of raw materials were adopted for feed formulation, not only to maintain the nutritional balance, but also to retain its palatability. Exogenous enzymes are good for the digestion of the larval diet (Tovar et al. 2002). Enzyme activity of the diet is correlated with temperature and pH, so enzymes are easily destroyed in many manufacturing processes. Papain and fodder yeast were added to the basal diet in this study, which avoided destroying the enzymes during the diet preparation process. Many studies with different fish species conclude that larvae could synthesize an appropriate amount of enzymes. However, the mechanism via which exogenous enzymes acts is not clearly understood. The coact of exogenous enzymes and endogenous enzymes to digest feed needs further research. Supplementation of artificial feeds with algal extracts has been demonstrated to enhance trypsin activity and to promote growth and survival (Cahu et al. 1998). Ingestion of algae was necessary for optimal assimilation of the zooplankton component of the diet (Rodriguez et al. 1994). In our study, the Schizochytrium algal meal was included in the feed formulation.
Higher growth rates were significantly correlated with higher enzyme activity, suggesting that digestive enzyme activity could be used as an indicator of larval condition. Alkaline phosphatase and amylase activity exhibited the best correlations with growth rate. A significant correlation between enzymatic digestive capacity and growth rate and food intake was found in cod larvae (Lemieux et al. 1999). Digestive enzymes were affected by feeding behavior and the biochemical composition of the food, as well as by a number of other factors. Although factors such as genetic control, gut morphology, and trophic level act to constrain the enzymatic response to diet, abundant evidence indicates that enzymes were modulated depending on diet composition, at least during late larval stages. Effects of different diets on digestive enzymes activity of early postlarvae Litopenaeus vannami were studied (Brito et al. 2000). This suggests that enzyme measurements reflect more clearly the feeding conditions during the early life of larvae than conventional survival and growth parameters (Cahu & Zambonino Infante 1994). The change from an Artemia diet to a microdiet influenced the digestive enzyme activity in the postlarvae. The digestive enzymes of larvae not only estimate the artificial feed and live food, but also speculative the reasonable of the formula of feed. However, the adaptation of digestive enzymes to feed is not well known and, often, contradictory results have been obtained (Le Moullac et al. 1996). Digestive enzymes in goldfish larvae showed a different level of activity when fed a compound diet or live prey (Abi-Ayad & Kestemont 1994). However, there was little information about different types and amount of binder influence on the digestive enzyme of the larvae. Different aspects of research should be addressed with the incorporation of the ontogenetic development of larvae and juvenile digestive systems, direct or indirect activation of digestive enzymes, and digestibility of microdiet ingredients, especially proteins, binders, and capsules ingredients. In our study, digestive enzymes greatly depended on the feed composition. In group II and group III, 92.2% feed composition was the same, and the enzyme activity of the larvae in the 2 groups confirmed that the digestive enzymes adapted to the feed composition. In the control group and group I, the significant difference in growth of the larval shrimp and unknown nutrients in the shrimp flake led to difficultly in comparing digestive enzyme activity.
Trypsin is the only pancreatic enzyme that can activate other digestive enzymes, and thus it may play an essential role in the digestive process of fish larvae. Trypsin activity has been detected during larval development in most studied marine fish species, and its variations coincide with the start of exogenous feeding, changes and origin of food, and digestive system maturation. Fodder yeast and Schizochytrium algal meal were constituents of the diet formulation and might have affected the trypsin activity, resulting in the lower trypsin secretion. Trypsin activity could increase as a consequence of an adjustment to a low protein content in the diet. Variations in trypsin activity probably occur because different types of dietary protein require different types of enzymes for its digestion. In current study, the highest trypsin activity was observed in the larvae fed the diet microencapsulated with ethyl cellulose, and this result coincided with the highest growth rate of the larvae in group III.
An adaptation of amylase to the high starch content in the diet has been described for sea bass larvae (Peres et al. 1996). The decline in amylase activity was normally delayed by dietary starch during larval development. Similar or higher levels for trypsin and amylase activity were observed in the pancreas of European sea bass larvae reared on a microparticulate diet compared with those reared on live prey (Cahu & Zambonino Infante 1994). In the current study, there was no significant difference in amylase activity between group II and group III. Two different wall materials induced no profound change in amylase activity. The highest amylase activity was found in group I, and this may be the result of the fact that many kinds of feed stimulated the secretion of the amylase enzyme.
Alkaline phosphatase is involved in the mechanisms of absorption and transport across the intestinal epithelium, and its activity is modulated by the quantity of diet ingested. The higher alkaline phosphatase activity suggested a better developmental and nutritional status in Artemia than in fed groups (Ribeiro et al. 2002). In the current study, diet had no effect on alkaline phosphatase activity. In a sense, the larvae in all groups exhibited a good nutritional status.
Gelatin is a protein and ethyl cellulose is a kind of cellulose. In this study, the cellulose activity of larval shrimp was not tested, because of the trace amount present. It is unknown whether wall material influences cellulose activity and other enzyme activity. It must be mentioned that the digestive enzyme activity of the larvae cannot reflect the ability of digestion and absorption of the nutrients. Developing a microdiet for fish larvae is more complicated than just finding the right combination of nutrients. Many drawbacks to a microdiet are still evident today and include visual attractiveness, palatability, high rates of leaching, and the need to measure digestibility quantitatively to evaluate progress. In conclusion, the microdiet can substitute for live food, which largely depends on the development of the microdiet and the larval nutrition.
We thank Fenglin Gu and Zhe Li for their assistance with this study.
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ZHONGGUO XIE, FURONG WANG, AIXIA ZHU, HUAXIN NIU, HAIYING LIU AND SHIDONG GUO *
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Lihu, Wuxi, Jiangsu 214122, PR China
* Corresponding author. E-mail: email@example.com
TABLE l. Proximate analysis (measured in grams per 100 g dry matter) of the diets mieroeneapsulated with different wall materials. Wall Material Moisture Crude Protein Crude Lipid Ash Gelatin 8.49 51.92 11.05 10.07 Ethyl cellulose 5.98 49.13 12.81 10.32 Partial data are published in Xie et al. (2010). TABLE 2. Frequency distribution of diameter and bulk density of the diets microencapsulated with gelatin and ethyl cellulose. Gelatin Ethyl Cellulose Wall Material Bulk Density Bulk Density Diameter ([micro]m) % (g/100 mL) % (g/100 mL) <150 1.6 44.6 7.9 42.7 150-178 4.7 42.2 14.7 40.0 178-250 12.0 39.5 22.7 38.2 250-420 23.7 37.3 27.8 36.2 420-590 29.6 36.1 19.3 33.8 >590 28.7 34.9 7.5 32.1 Partial data are published in Xie et al. (2010). TABLE 3. Inclusion, encapsulation, and retention efficiency of the diets microencapsulated with different wall materials. Inclusion Encapsulation Wall Material Efficiency (%) Efficiency (%) Gelatin 92.2 [+ or -] 1.6 76.8 [+ or -] 4.1 Ethyl cellulose 95.8 [+ or -] 1.2 85.3 [+ or -] 3.5 Retention Wall Material Efficiency (%) Gelatin 60.6 [+ or -] 5.2 Ethyl cellulose 75.5 [+ or -] 4.7 Partial data are published in Xie et al. (2010). TABLE 4. The pH value of the microencapsulated diets in different solution. Distilled 35"NaCl Seawater, Wall Material Water Solution pH 8.2 Gelatin 5.98 5.76 7.82 Ethyl cellulose 6.24 6.17 7.97 TABLE 5. Growth performance and body composition (dry matter) of the larvae (Penaeus japonicus) fed different diets. Wet Total Weight Length Treatment (mg) (mm) Control 3.04 [+ or -] 0.07 (a) 9.90 [+ or -] 0.20 (a) Group I 3.38 [+ or -] 0.03 (b) 10.73 [+ or -] 0.15 (ab) Group II 3.88 [+ or -] 0.05 (c) 11.13 [+ or -] 0.15 (b) Group III 4.01 [+ or -] 0.13 (c) 12.20 [+ or -] 0.98 (c) Crude Crude Survival Protein Lipid Treatment (%) (%) (%) Control 71.43 [+ or -] 4.67 (a) 67.83 9.32 Group I 72.53 [+ or -] 1.05 (a) 66.92 9.86 Group II 84.33 [+ or -] 1.58 (b) 70.94 10.69 Group III 85.64 [+ or -] 2.90 (b) 69.38 10.16 (a,b,c) Means in the same row with no common superscripts differ significantly (P < 0.05). Partial data are published in Me et al. (2010). TABLE 6. Digestive enzyme activity (units per milligram protein) of the larvae (Penaeus japonieus) fed different diets. Treatment Trypsin Amylase Control 1.61 [+ or -] 0.09 (a) 0.503 [+ or -] 0.033 (a) Group I 2.31 [+ or -] 0.20 (b) 0.664 [+ or -] 0.055 (b) Group II 2.13 [+ or -] 0.21 (b) 0.622 [+ or -] 0.054 (b) Group III 2.67 [+ or -] 0.11 (c) 0.641 [+ or -] 0.041 (b) Alkaline Trypsin/ Treatment Phosphatase Amylase Control 0.076 [+ or -] 0.014 (a) 3.20 Group I 0.087 [+ or -] 0.009 (a) 3.47 Group II 0.091 [+ or -] 0.006 (a) 3.42 Group III 0.092 [+ or -] 0.007 (a) 4.16 (a,b,c) Means in the same row with no common superscripts differ significantly (P < 0.05).
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|Author:||Xie, Zhongguo; Wang, Furong; Zhu, Aixia; Niu, Huaxin; Liu, Haiying; Guo, Shidong|
|Publication:||Journal of Shellfish Research|
|Date:||Apr 1, 2011|
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