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ScienceDirect Aquatic Procedia 7 (2016) 46 – 53

2nd International Symposium on Aquatic Products Processing and Health ISAPPROSH 2015

Effect of Phytase Enzyme on Growth Boost in the Artificial Feed Made of Plant Protein to Shorten Production Time of Giant Tiger Prawn [Penaeus monodon, (Fabricus 1798)] Diana Rachmawati*, Istiyanto Samidjan Department of Fisheries, Faculty of Fisheries and Marine Sciences, Diponegoro University, Jl. Prof. Soedarto, SH, Tembalang, Semarang, 50275, Jawa Tengah, Indonesia

Abstract The use of plant protein in the artificial feed needs to be reckoned since plant protein contains phytate acid. To solve the problem is by adding phytase enzyme. This study aims to identify effect of phytase enzyme in the artificial feed and determine optimal dose of phytase enzyme on feed digestibility, nutrient efficiency utilization, and growth of giant tiger prawn (Penaeus monodon). The shrimp used in the study was giant tiger prawn (P. monodon) with the average weight of (1.19 ± 0.06) g per shrimp and the density of one shrimp per L. Methodology used in this study was experimental treatments with complete random design. The study consisted of four treatments and three repetitions. The treatments were by adding phytase enzyme in the different doses, namely: A (0 FTU · kg–1 diet), B (500 FTU · kg–1 diet), C (1 000 FTU · kg–1 diet) and D (1 500 FTU · kg–1 diet). Data collected were from variables of digestibility raw protein (DRP), digestibility total protein (DTP), nutrient efficiency utilization (NEU), relative growth rate (RGR), survival rate (SR) of giant tiger prawn (P. monodon) and water quality. The results show that the treatments significantly (p < 0.01) affected on the DRP, DTP, NEU, and RGR; however, they did not significantly (p > 0.05) influence on the survival rate. The optimum dose of phytase enzym for feed digestibility, feed utilization and the growth of giant tiger prawn (P. monodon) was 1 000 FTU · kg–1 feed. The water quality was still in the viable range for giant tiger prawn cultivation. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license © 2016 The Authors. Published by Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-reviewunder under responsibility of science the Science and Editorial of ISAPPROSH 2015. Peer-review responsibility of the and editorial board ofBoard ISAPPROSH 2015 Keywords: Artificial feed; tiger prawn (Penaeus monodon, Fabricus 1798); growth; phytase enzyme; production

* Corresponding author. Tel.: +62 813 132 8354;fax :+62 024 747 4698 E-mail address: dianarachmawati1964@gmail.com

2214-241X © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the science and editorial board of ISAPPROSH 2015 doi:10.1016/j.aqpro.2016.07.006

Diana Rachmawati and Istiyanto Samidjan / Aquatic Procedia 7 (2016) 46 – 53

1. Introduction Giant tiger prawn (Penaeus monodon, Fabricus 1798) is an endogenous species of Indonesia. The popularity has faded since the introduction of vannamei shrimp which is able to live in more dense population per unit area, has more disease resistant, and has higher sale price. The success of giant tiger prawn cultivation depends on the artificial feed and disease outbreak. One of the ingredients in the artificial feed is soybean meal, but unfortunately it contains anti-nutrient, phytate acid (Kumar et al., 2011). Cao et al. (2007) has reported that phytate acid in the soybean meal was 3.88 g · kg–1. Phytate acid bind minerals which have valence of two or three (calcium, ferrum, zinc, magnesium) to form compound is difficult to digest (Baruah et al., 2007). Besides binding with minerals, phytate acid also binds with protein and amino acid; therefore it reduce feed digestibility (Ravindran, 2000). One way to mitigate the problem was by adding phytase enzyme (Chung, 2001), and this idea was supported by Jobing (2002) and NRC (1983) that to reduce phytate acid content can be used phytase enzyme. The enzyme in the artificial feed can improve nutrient absorption and regulate nutrient excretion, such as phosphor, nitrogen, and mineral, and hydrolyze phytate acid to become inositol and phosphate acid. Hydrolyzation can break the minerals from the compound (Chung, 2001). Moreover, Baruah et al. (2007) found those phytase enzymes can hydrolyze phytate acid (mio-inositol hexaphosphat) into mio-inositol mono, di, tetra, and pentaphosphate and organic phosphate. Some studies on the effect of phytase enzyme in the artificial feed were conducted by Debnath et al. (2005), Baruah et al. (2004), Rachmawati and Hutabarat (2006), Suprayudi et al. (2012), Shapawi et al. (2003), Bulbul et al. (2015), and Danwitz et al. (2016). Debnath et al. (2005) studied that the addition of phytase enzyme as much as 500 FTU · kg–1 diet can increase growth of Pangasius pangasius (Fawler, 1937) fingerings, while Baruah et al. (2004) found that the additional phytase enzyme of 750 mg · kg–1 soybean meal can improve growth and digestibility of Labeo rohita (Hamilton, 1822). Rachmawati (2006) and Hutabarat (2010) also discovered that the addition of 1 000 unit of phytase enzyme · kg–1 soybean meal can increase growth of Epinephelus fuscoguttatus (Forsskal, 1775) and Osphronemus gouramy (Lacepede, 1801). The additional pyhtase enzyme of 500 unit · kg–1 soybean can increase phosphor digestibility and growth of Lipopenaeus vannamei (Boone, 1931) (Suprayudi et al., 2012). Shapawi et al. (2013) detected that and 200 mg · kg–1 30 % soybean meal of diet can improve nutrient digestibility and growth of kuruma shrimp (Marsupenaeus japonicas, Bate 1888), meanwhile Danwitz et al. (2016) mentioned that the addition of phytase enzyme of 2 000 FTU · kg–1 diet can increase growth, protein and phosphor digestibility of turbot fish (Psetta maxima, Linne 1758). This study aims to identify effect of phytase enzyme in the artificial feed and determine optimal dose of phytase enzyme on feed digestibility, nutrient efficiency utilization, and growth of giant tiger prawn (P. monodon). 2. Materials and methods Shrimp used in the study was giant tiger prawn (P. monodon) with the average weight of (1.19 ± 0.06) g per shrimp. Experimental shrimp was selected based on the homogeneity, completeness of body parts, and overall healthiness. To adapt to the feed and the new environment, experimental shrimp was put in the media for one week. And the shrimp was allowed to fast for one day to give time for excretion of the metabolism waste (Rachmawati and Hutabarat, 2006). The growth for the samples was measured every week. The media used in the experiment was 12-black buckets with the size of 25 L each. The buckets were disinfected using kalium permanganat (K2PO4) and sunbathed until dry. Then the buckets were arranged randomly and poured with 20 L water each. After filling the buckets with the water, they were aerated and covered with clear plastics as biosecurity. Feed used in the study was in the form of pellet with the size of 0.2 mm to 0.5 mm. The feed contained minimum of 38 % protein and 3 200 kcal DE · kg–1 (Suprayudi et al., 2012). Shrimp was fed at satiation four times a day at 07:00, 11:00, 15:00, and 19:00 a clock. The feed made of fish meal as a source of animal protein, soybean meal as a source of plant protein, corn meal, rice bran, and wheat flour as a source of carbohydrate, fish and corn oil as sources of fat, minerals and vitamin mix (aquamin) as sources of vitamin, CMC as a binder, 1 % of Cr 2O3 as an indirect indicator of digestibility (NRC, 1993) and phytase enzyme as a breaker phytate acid compound. The brand of phytase enzyme used in the study was Natuphos® 5000 produced by PT. BASF Indonesia. Composition of

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artificial feed can be seen in the Table 1. Table 1. Composition and proximate analysis in the artificial feed Ingredients

A

B

Composition C 1 000 28.00 37.80 6.70 8.88 12.00 3.00 2.00 1.00 1.00 1.00

D Phytase Enzyme (FTU) 0 500 1 500 Fish meal 28.00 28.00 27.90 Soybean meal 37.80 37.80 37.80 Corn meal 6.50 6.70 6.80 Rice bran 7.90 8.89 8.90 Wheat flour 13.00 11.90 11.99 Fish oil 2.99 3.00 2.90 Corn oil 2.00 2.00 1.99 Vitamin&Mineral Mix 1.00 1.00 1.00 CMC 1.00 1.00 1.00 Cr2O3 1.00 1.00 1.00 Proximate Analysis Protein (%)* 38.12 38.13 38.14 38.09 Fat (%)* 10.76 10.64 10.66 10.53 BETN (%)* 34.34 34.34 34.40 34.50 Energy (kcal)** 306.42 305.49 305.80 304.89 Ratio E/P 8.04 8.01 8.02 8.00 Notes: A (0 FTU · kg–1 diet), B (500 FTU · kg–1 diet), C (1 000 FTU · kg–1 diet), D (1 500 FTU · kg–1 diet) (i). The values were calculated based Digestible Energy (Wilson, 1982) for 1 g protein equals 3.5 kcal, 1 g fat equals 8.1 kcal, and 1 g carbohydrate equals 2.5 kcal. (ii). According De Silva S.S., Perera, M.K. (1987), the optimal E/P ratio for growth ranges from 8 kcal · g–1 to 12 kcal · g–1. (iii) *Animal Nutrient Laboratory, Faculty of Husbandry and Agriculture, Diponegoro University (2015) ** The SI derived unit for energy is the joule (1 kcal = 4.1868 kJ)

Experimental randomized complete design was used with five treatments and three repetitions. The treatments in this study were to add various doses of phytase enzyme, namely A (0 FTU · kg–1 diet), B (500 FTU · kg–1 diet), C (1 000 FTU · kg–1 diet), D (1 500 FTU · kg–1 diet). The treatments used in this study was modified from Suprayudi et al. (2005) who reported that the enzyme dose of 750 FTU · kg–1 diet resulted in the best growth and diet conversion for vanname shrimp with the weight of fingerling of (3.8 ± 0.01) g. Measurement of the variables on digestibility raw protein (DRP), digestibility total protein (DTP), nutrient efficiency utilization (NEU), relative growth rate (RGR), survival rate (SR) of giant tiger shrimp (P. monodon) and water quality consisting of temperature, pH, Dissolved Oxygen (DO) and salinity was conducted every day, while ammoniac level was measured at the beginning and end of study. Analysis of Variance was used to analyze the data. If the results analysis of variance indicated significant (p < 0.05) or very significant (p < 0.01), then double area Duncan test to determine the difference of the mean was conducted. The next step was to find out the optimum dose of phytase enzyme using Polynomial Orthogonal test. Statistical software used for the analyses were SAS version 9.0 and Maple version 12.0 (Steel and Torrie, 1993). Descriptive analysis was used to explain water quality. 3. Results and discussions The results of the study were shown in the Table 2. Table 2. The mean of variables Data

Treatments

DRP (%) DTP (%) NEU (%)

A 70.75 ± 0.58c 50.89 ± 0.70c 29.08 ± 1.50c

B 75.86 ± 0.60bc 58.90 ± 0.63bc 32.29 ± 0.58bc

C 85.86 ± 0.60a 68.20 ± 0.45a 37.38 ± 1.97a

D 78.35 ± 0.09a 60.64 ± 0.60a 36.44 ± 2.70a

RGR (% per day) SR (%)

1.94 ± 0.20bc 86.67 ± 5.77a

2.41 ± 0.13ac 93.33 ± 5.77a

2.97 ± 0.23a 93.33 ± 5.77a

2.76 ± 0.14a 93.33 ± 5.77a

Note: Values ± SD in the same superscript in a column show no significant difference


The measurement results of the water quality which was used as cultivation media for giant tiger prawn (P. monodon) were depicted in the Table 3. The water quality measurement show that the water quality during experiment was still in the viable range for giant tiger prawn (P. monodon). Table 3. The results of water quality measurement Treatments A B C D Values Note: FAO (1988)

Temperature (ºC) 26 to 32 26 to 32 26 to 32 26 to 32 23 to 33*

pH 6.8 to 7.3 6.8 to 7.3 6.8 to 7.3 6.8 to 7.3 7 to 8.5 *

Range of Water Quality DO (mg · L–1) 4.03 to 5.5 4.04 to 5.6 4.06 to 5.6 4.03 to 5.15 < 3*

Ammoniac Total (mg · L–1) 0.000 to 0.030 0.000 to 0.028 0.000 to 0.022 0.000 to 0.025 < 0.10 *

The addition of phytase enzyme in the artificial feed influenced significantly (p < 0.01) on digestibility raw protein and digestibility total protein of giant tiger prawn, as shown in the Table 2. The additional phytase enzyme as much as 500 FTU · kg–1 to 1 500 FTU · kg–1 diet can increase digestibility raw protein and digestibility total protein of giant tiger prawn that is consistent with the finding by Storebakken et al. (1988). It has been found that the addition of phytase enzyme can improve protein digestibility and protein retention. Moreover, Hunter (2001) also reported that the addition of phytase enzyme in the artificial feed can increase protein digestibility from 84.5 % to 87.7 %. Similar results were also found in the carp (Vielma et al., 2004), rainbow trout (Sugiura et al., 2001; Forster et al., 1999), Labeo rohita (Husain et al., 2014). Moreover, Baruah et al., (2004) have observed that the addition of the phytase enzyme in the artificial feed made of plant ingredients increased protein digestibility due to the breakdown of phytin-protein compounds. The highest digestibility raw protein and digestibility total protein of giant tiger prawn (P. monodon) were obtained from the additional phytase enzyme of 1 000 FTU · kg–1 diet (C treatment), meanwhile the lowest happened in the addition of the enzyme of 0 FTU · kg–1 diet (A treatment). The high digestibility in the treatment C was thought due to the appropriate dose of phytase enzyme to effectively break down anti-nutrient and to increase diet digestibility, as reported by Cao et al. (2007) that phytase enzyme was able to break down anti-nutrient, such as phytate acid, non-starch polysaccharide, trypsin inhibitor, and to increase diet digestibility so it can raise nutrient level. Debnath et al. (2005) also mentioned that protein utilization and digestibility in the Atlantic salmon were significantly boosted by the addition of phytase enzyme in the artificial feed, otherwise the digestibility was low it the phytate enzyme did not present. Table 2 shows that various doses of phytase enzyme in the artificial feed gave very significant effect (p < 0.01) on the nutrient efficiency growth of giant tiger prawn (P. monodon). It was expected due to the ability of phytase enzyme to increase the effectiveness and efficiency of energy use. Phytate acid compound in the feed that inhibited nutrient digestibility in the intestinal system can be hydrolyzed (Wang et al., 2014). The efficiency values of nutrient utilization was 68.38 % ± 2.24 % at the dose of 1 000 FTU · kg–1 diet. This values were lower than that of Suprayudi et al. (2012) findings in the vanname shrimp (L. Vannamei), that were 73.06 % ± 4.59 % at the dose of 500 FTU · kg–1 diet. Different nutrient utilizations were thought due to the difference of fish species; therefore, they have different growth rates. Montgomery (2010) findings supported the view as he mentioned that to achieve subadult phase of giant tiger prawn (P. monodon) needs 5 mo to 6 mo, while vanname shrimp (L. vannamei) to get the same phase needs only 3 mo to 4 mo. High nutrient efficiency reflected that small amount of nutrient was converted into energy, while the big amount of nutrient was used to grow (NRC, 1993), as found in this study that the highest daily growth was (0.97 ± 0.23) % per day and the highest value of nutrient efficiency utilization was 37.38 % ± 1.97 %, both reached at the dose of 1 000 FTU · kg–1 diet. The Orthogonal Polynomial test was used to determine optimum dose of phytase enzyme to add to artificial feed on EPP of the giant tiger prawn (P. monodon). It had cubical relationship with the equation y = - 418.9x3 + 389.7x2 – 29.93x + 54.45 and R2 = 0.83, as shown in the Figure 1. The optimum dose was 1 079 FTU · kg–1diet which could result in the highest NEU of 68.38 % per day.

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Figure 1. Graph of orthogonal polynomial of nutrient efficiency utilization (%/day) in the giant tiger prawn (P. Monodon).

Various doses of phytase enzyme in the artificial feed resulted in the significant effect (p < 0.01) on the relative growth of the giant tiger prawn (P. monodon). It was thought that phytase enzyme can break down complex compound of phytate acid in the artificial feed which the phytate acid was anti-nutrient inhibiting nutrient absorption, including protein which is an important growth ingredient, in the intestinal system (Xue, 2014). Chung (2001) also reported that phytase enzyme can hydrolyze phytate acid into inositol and phosphate acid, so it can improve nutrient absorption and regulate nutrient excretion, such as phosphor, nitrogen, and minerals. Optimum nutrient absorption can increase shrimp growth. Relative growth rate is closely related to nutrient efficiency utilization. The more efficient in the nutrient utilization is the more efficient in the protein utilization; therefore, the availability of protein in the artificial feed is more abundant for the giant tiger prawn (P. Monodon) growth. This was similar to Baruah et al. (2004) finding that the addition of phytase enzyme can reduce the content of phytate acid in the artificial feed made of plant protein and increase relative growth rate and nutrient efficiency utilization. Baruah et al. (2007) and Fox et al. (2006) also found out that the addition of phytase enzyme in the artificial feed made of plant source for rainbow trout can increase growth, coefficiency of raw protein, total phospoor, and phytatephosphor. The phytase enzyme dose of 1 000 FTU · kg–1 diet (C treatment) gave the highest relative growth rate of (2.97 ± 0.23) % per day. It was thought that the enzyme can unbind phosphor, protein, and mineral from soybean meal as reported by Tahoun et al. (2009). He found that the unbinding complex compound can ease phosphor, protein, and mineral absorption to optimize the growth. Similar results were also reported by Weerd et al. (1999) for African catfish [Clarias gariepinus (Burchell, 1822)], Papatryphon et al. (1999) for Stripped bass [Morone saxatilis (Walbaum, 1792)], Vielma et al. (2004) for Onchorhynchus mykiss (Neave, 1944), Sajjadi and Carter (2004) for Atlantic salmon [Salmo salar (Linnaeus, 1758)], and Yoo et al. (2005) for Sebastes schlegeli (Hilgendorf, 1880). The lowest relative growth rate was obtained when there was no addition of phytase enzyme (A treatment) with the growth of (1.94 ± 0.20) % per day. It was because of phytase enzyme absence to hydrolyze phytase acid. This finding was supported by the study of Rachmawati and Hutbarat (2006) that the treatment without phytase enzyme resulted in low utilization of protein and complex compound. Fox et al. (2006) also suggested that unbinding phytate acid would bind protein and phosphor, so they were difficult to absorb. Similar results were found in other species (Pongmaneerat and Watanabe, 1992; Rumsey et al., 1994; Stickney et al., 1996; Yoo et al., 2005; Hassan et al., 2009). The laboratory results for phytate acid contents in the A, B, C, and D treatments were 0.75 %, 0.72 %, 0.70 %, and 0.68 % respectively. The presence of phytate acid could cause a growth decrease as reported by NRC (1993). NRC reported that 0.5 % phytase enzyme in the feed can decrease growth and nutrient efficiency in the rainbow trout (O. mysskis). Tacon (1995) also mentioned that 2.58 % phytase enzyme in the feed could cause growth, nutrient efficiency, and protein efficiency to decrease and cause mortality. Alvi (1994) reported that the specific


growth in the Labeo rohita fish has decreased when the level of phytate acid is more than 1 %, on the other hand the growth increased when phytase enzyme is added into artificial feed. The laboratory results for phytate acid contents in the feces of the A, B, C, and D treatments were 0.60 %, 0.55 %, 0.38 %, and 0.46 % respectively. From those information, the values of phytate acid decrease were 0.15 %, 0.17 %, 0.32 %, and 0.22 % respectively for the treatment of A,B,C, and D. These indicated that phytate acid has been broken down by the phytase enzyme. It could boost nutrient absorption (Winarno, 1987). The measurement on the optimum dose for relative growth in the giant tiger prawn (P. monodon) used Polynomial Orthogonal test and it resulted in the cubical equation Y = -143.9x3 + 47.841x2 + 1.3208x + 1.9434 with the R2 = 0.87, as shown in the Figure 2. Using the equation optimum dose could be calculated. The optimum dose was 1 035 FTU · kg–1diet and gave maximum relative growth of 3.03 % per day.

Figure 2. Graph of relative growth rate in the giant tiger prawn (P. monodon)

Various phytase enzymes in the artificial feed did not significantly influence on the survival rate of giant tiger prawn (P. monodon). The results were in accordance with the study by Robinson et al. (2002) that stated survival rate was insignicantly influenced by the addition of phytase enzyme. Some factors that affected survival rates are biotic factors and abiotic factors, (Hepher, 1988). Biotic factors include competitiveness, parasites, age, predators, density and management, while abiotic factors include physic and chemical characteristics physic and chemical characteristics physic and chemical characteristics. The survival rate in the tiger prawn (P. monodon) in this study ranged from 86.67 ± 5.77 to 93.33 ± 5.77 and they were considered high. This high survival rate indicated that the quality and quantity of enough feed to fulfill the needs. Good feed is the feed that contains balanced nutrients and does not poison the shrimp. Balanced protein is very important in the diet formulation because it has a big role in growth and disease resistance. Internal and external factors were also important factors on growth and disease resistance (Suprayudi et al., 2012). 4. Conclusion x x

Phytase Enzyme in the artificial feed has very significant effect on nutrient digestibility, nutrient efficiency utilization, relative growth rate, but it was insignificant to survival rate in the giant tiger prawn (P. monodon). 1 000 FTU · kg–1 diet was the best dose of phytase enzyme on nutrient digestibility, nutrient efficiency utilization, and survival rate in the giant tiger prawn (P. monodon).

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