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Home>FFTC Document Database>Extension Bulletins>The Profile of Essential Amino Acid, Fatty Acid and the Growth of Chaetoceros gracilis Using Different Technical Media Guillard and Double Walne
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Vivi Endar Herawati1, Johannes Hutabarat1,
Slamet Budi Prayitno1, Y.S. Darmanto2

1Department of Aquaculture, Faculty of Fisheries and Marine Science,
Diponegoro University Jl. Prof Soedharto, Semarang 50275, Indonesia

2Department of Food sciences; Faculty of Fisheries and Marine Science;
Diponegoro University Jl. Prof Soedharto, Semarang 50275, Indonesia

Email: anshinvie@yahoo.com

ABSTRACT

The objective of this research is to discover the difference between the dissimilar technical culture media (Walne dan Guillard) in Chaetoceros gracilis upon the growth, fatty acid profile, and essential amino acid profile. The culture method is used massively upon two dissimilar technical culture media (Double Walne and Technical Guillard). The analysis of the fatty acid is performed using the transesterification in situ method, and that of the essential amino acid profile using HPLC Eurospher 100-5 C18, 250x4.6mms. The research result shows that Technical Guillard makes the better culture media, because the growth of Chaetoceros gracilis reaches 54.60 x 104 cell/ml in a 4-day stationary phase. The Technical Guillard media results in the better fatty acid profile and essential amino acid of Chaetoceros gracilis compared to the other media: in Chaetoceros gracilis the palmitic fatty acid is 15.30%, oleic 25.57%, linoleic 6.38%, linoleic 3.77%, EPA 8.16. The higher domination of Chaetoceros gracilis in the essential amino acid resides in the Threonim amino acid: 2,359.05 ppms in Chaetoceros gracilis.

Keywords:     Chaetoceros gracilis, Technical culture media  Guillard and Double Walne, Growth, Proksimat, Fatty Acid Profile and Essential amino Acid 

INTRODUCTION

Natural feed as a helper for fish cultivation and a supporting factor for the success of the cultivation has been industriously cultivated (Herawati, 2008). The natural feed itself is the living feed which is obtained from nature. Chaetoceros gracilis, one of species diatoms which become natural feed for fish larvae and the diatoms are primely edible for prawns (Herawati, 2013). The edibility is caused by the diatoms' nutrient content, and the size of Chaetoceros gracilis which fits suitably in the mouth opening of mainly prawn larvae from the naupli to mysis stadium. Chaetoceros gracilis are single-celled organism; according to Meyer (2010), C. calcitrans have 7.3±0.8 µm diameter and 4.9±0.3 µm length. The diatoms are superior for their high nutrient value with protein content of approximately 21.63 _ 32.05% (BBPBAP, 2010 and Herawati, 2013). The need of mainly prawn larvae for Chaetoceros gracilis as their natural feed is large; in order to fulfill the need for natural feed a massive-scale culture of Chaetoceros gracilis is necessary for sustainability in hatchery operations of mainly prawn.

The need for Chaetoceros gracilis as a natural feed in the seeding activity is huge, so a massive culture with high density in a short period of time is necessary. In addition to producing high cell abundance, the massive culture is expected to produce maximum nutrient content through the fatty acid profile and essential amino acid.

In order to have optimum growth and nutrient, a proper media is necessary. The culture media which are often utilized to culture diatoms are Guillard (Anderson, 2005; BBPAP, 2007; Amsler, 2008) and double Walne (Rousch, 2003; Susanto dkk., 2006 ; BBPAP, 2010). Modification of media Walne (double Walne and silicate addition) is able to increase the cell number growth of Chaetoceros gracilis 3-4 times (Susanto et al., 2006) and spur the growth 4-5 times (BBPAP, 2010). This research profoundly studies the use of different technical culture media (modified Walne and Guillard) upon the growth, essential amino acid profile and fatty acid profile of massive cultured Chaetoceros gracilis.

The objectives of this research are to figure out the difference between the technical culture media double Walne and Guillard upon the growth of Chaetoceros gracilis and to figure out the fatty acid profileand essential amino acid of Chaetoceros gracilis in the two different technical media.

METHODOLOGY

Materials and tools

The materials which are utilized in this research are the seeds of Chaetoceros gracilis. The test culture media which are utilized are Walne fertilizer (Anderson, 2005) which is composed of double Walne with silicate addition (BBPAP, 2007) and technical Guillard (Anderson, 2005). The tools which are utilized in this research are autoclaph, haemocytometer, 2 ton volume fibre bath, lux meter, water quality checker, microscope, planktonet, rocks, aeration rocks and hoses, elemeyer, HPLC Eurospher 100-5 C18, 250x4,6mms and GC W COT Fused Siliate Coating CP-SIL-88 Tailor Made Fame for fatty acid analysis.

Diatom seeds

The diatom seeds were obtained and cultured purley from Laboratorium Pakan Alami BBPBAP (Natural Feed Laboratory) in Jepara. Before the seeds of C. gracilis were planted, the seeds were aerated for 15 minutes so that they would not produce sediments. The seeds of Chaetoceros gracilis were then planted with initial density of 50, 000 cell/ml.

Culture media

The culture media which were utilized in this research were double Walne dan technical Guillard culture media to culture massively Chaetoceros gracilis The culture media are presented in Table 1.

The media water which was utilized to culture was prepared with a salinity of 25-28 ppt (Abdulgani et.al., 2008). The environment culture is presented in Table 2.

Culture practice

The research culture practice was carried out by making the media, mixing the materials, except for the vitamin; the mixture was then stirred until it became homogenous. The media was, later on, autoclaved for two hours. The media volume of treatment test was counted individually of 1, 500 litre; each treatment comprised of the micture of fertilizer and sterilized sea water.

The number of seeds which is expected for the initial stocking is counted by using the following formula of seed dilution (Amsler, 2008):

  • V1x N1=V2xN2
  • Information:
  • V1= Volume of seeds which is required for the initial stocking
  • V2 = Volume of media water which is to be stocked with seeds
  • N1 = The number of Chaetoceros sp stock
  • N2 = The number of Chaetoceros sp which is expected.

After the initial density was figured out, the culture stock solution was put into the 1 litre media in order to obtain the initial cell density of 50, 000 cell/ml. The density was counted by using Haemocytometer; the observation was carried out every 24 hours for the growth of C. gracilis.

The harvesting of C. calcitrans was carried out by lifting up the aeration into idleness. After C. calcitrans sediments, the sediment was ciphoned. The ciphone of C. calcitrans was filtered by using planktonet so as to obtain the harvesting of C. gracilis in the form of pasta.

Fatty acid analysis

The fatty acid analysis by using GC follows the procedure which is conducted at Laboratorium Tekhnologi Pangan (Food Technology Laboratory) of GajahMada University, Yogyakarta, and it also follows that of Park and Goins (1994).

100 µl Chaetoceros calcitrans, being homogenated with 4 ml of distilled water, resulted in 100 µl homogenate. The samples were put in test tubes, to which 100 µl methyl chloride and 1 ml NaOH 0.5 N methanol were added. The test tubes were filled with nitrogen and sealed, and then the tubes were heated in temperatured water heater of 90ºC for 10 minutes. The tubes, later on, were cooled down and added with 1 ml BF3 14% methanol. The tubes were filled with nitrogen, and the heating is resumed at the same temperature for 10 minutes. Next, the tubes were cooled at the room temperature and added with 1 ml distilled water and 200 _ 500 µl hexane dan divortex for a minute to extract the fatty acid methyl esther. The flake, being decentrifuged, was hence ready for the GC analysis.

Essential amino acid analysis

The essential amino acid analysis, using HPLC, was conducted at Laboratorium Pengembangan dan Penelitian dan Pengujian Terpadu GajahMada (Gajah Mada Development and Research and Integrated Testing Laboratory); the analysis follows the procedure of LPPT Yogyakarta.

Samples were scaled at ± 2.5 grams; the samples were put in the sealed glass test tubes and were added with 15 ml HCl 6N divortex so as to become homogenous. The samples were then hydrolyzed by using the autoclaph at a temperature of 110ºC for 12 hours. The samples, later on, were cooled at a room temperature and neutralized using NaOH 6N. Next, the samples were added with 2.5 ml Pb acetate 40 % and 1 ml oxalic acid 15 % and were measured at 50.0 ml by using aquabidest. After that, ± 3 ml of the samples was taken by using millex filter 0.45; 25 µl + 475µL OPAA vortex soulution were injected to HPLC by using millex filter; and 30 µL soulution was to be reacted for three minutes before it was injected to HPLC.

Data analysis

The data analysis was conducted by using the T-test; the test was intended to figure out the difference between the culture treatment by using the double Walne and technical Guillard culture media upon the growth of Chaetoceros gracilis.

RESULTS AND DISCUSSION

Growth

One of the objetcives of algae culture is to obtain high cell abundance with optimum nutrient content (Fogg, 1965); the cell abundance is necessary because the natural feed inevitably needs to be given to fish and prawn larvae. The inevitability is caused by the irreplaceable nutrient content of the natural feed. The following graph shows the growth of Chaetoceros gracilis during this research.

The growth phase of C. calcitrans in the different technical culture media is presented in Table 3.

The statistic test result on the lag phase shows that double Walne and technical Guillard culture signicantly influences (P<0.05) upon the growth of C. gracilis; the significant influence was supposedly caused by the density of double Walne culture media which is almost similar to the cell fluid of C. gracilis's body. The close similarity makes C. gracilis in double Walne grow faster than that in technical Guillard culture media (Fig. 1); the lag phase occurred on the second day. Besides that, the statistic test result at the exponential phase shows that double Walne and technical Guillard culture media did not significantly influence (P>0.05) upon the growth of C. gracilis; the insignificant influence was supposedly caused by the density of the cell fluid of C. gracilis's body which is different from that of the culture media. The difference makes the cell of C. gracilis grow slower than that in the media; the exponential phase occurred on the third day. The statistic test result at the stationary phase shows that double Walne and technical Guillard culture media did not significantly influence (P>0.05) the growth of C. gracilis upon its highest density; the stationary phase lasted for four days.

Fatty acid profile

Table 4 presents the total fatty acid profile. The table shows that the concentration of the respective saturated fatty acid and unsaturated fatty acid Chaetoceros gracilis (46.27 % and 43. 88%) in technical Guillard culture media is higher than those in double Walne culture media: Chaetoceros gracilis (25.98 % and 20.79%). The total fatty acid profile is presented in Table 5.

The fatty acid profile shows that palmitate fatty acid is the highest in the saturated fatty acid column, and oleic fatty acid is the highest in the unsaturated fatty acid column. Palmitate fatty acid is a substrate in a biosynthesis of Safa fatty acid (Yap and Chen, 2001).

Essential amino acid profile

Table 6 presents essential amino acid profile. The table shows that the concentration of total essential amino acid Chaetoceros gracilis (701.09 ppm) in technical Guillard culture media is higher than those in double Walne culture media: Chaetoceros gracilis (515.42 ppm). The essential amino acid profile is presented in Table 5.

The analysis at the chromatogram shows that the content of essential amino acid in C. calcitrans is solely dominated by Threonine, resulting in 478.46 ppm for C. calcitrans in technical Guillard culture media, and 337.42 ppm for C.gracilis in double Walne culture media.

DISCUSSION

The growth of Chaetoceros gracilis in double Walne culture media is faster than that of the technical Guillard culture media because the density of double Walne is thicker than that of technical Guillard. Fogg's research (1965) states that the density of culture media influences the growth of microalgae: when there is no difference between the density of the culture media and that of microalgae, the growth of microalgae is faster than when there is some difference; the growth of microalgae takes longer time. The difference between the density of the culture media and that of the cell fluid in microalgae influences enzyme recovery, substrate concentration on the next growth level, and nutrient absorption through diffusion process (Harrison et al, 2008).

The analysis of fatty acid results in the fatty acid profile, with palmitate and oleic fatty acid as the highest fatty acid. The palmitate fatty acid functions to store energy in diatoms (Herawati, 2013). The research of Araujo (2010), concludes that the higher the palmitate fatty acid resides in the diatoms, the more energy in the diatoms which is usable as the energy for the cultivant consuming it. The diatoms contain a lot of oleic fatty acid; the oleic fatty acid is the building substrate for Pufa long chain fatty acid.

The oleic fatty acid functions as the basic substrate to build the Pufa long chain. The research of Roussch (2003), concludes that the oleic fatty acid is the substrate in denaturation process and catalytics extension. Biosynthesis of Pufa starts from the oleic fatty acid, then the linoleic fatty acid as the basic substrate for the building of omega 6 long chain; whereas the linolenic fatty acid functions as the basic substrate for the building of omega 3 long chain. The building of Pufa involves a series of denaturation process and catayltics extension, which is supported by desaturated and longased enzymes.

The amino composition of diatoms' protein among species is highly similar (Brown, 2002), and the composition is beyond the influence of the growth phase and light condition. The research result shows that the highest amino acid of C. gracilis resides in technical Guillard culture media. Brown's research (2002) reveals that the density of diatoms ranges from approximately 1.83 ppm _ 2.21 ppm; the research results in the density of 1.77 ppm for C. gracilis.

The chromatogram shows that threonine is the highest of its amino acid. Threonine amino acid functions to bond metal ions which are necessary for enzymatic reaction. Besides that, threonin also functions to aid the prevention of fat accumulation (Herawati, 2013).

The chromatogram, moreover, shows that valine amino acid is bound to thryptophan amino acid; thryptophan is the essential amino acid which has aromatic branched chains, and valine is the essential amino acid which has aliphatic branched chains. These two amino acids are bound through one peptide bond by releasing water molecules. The bond is often called as the balance reaction; the reaction is majorly directed to hydrolysis which does not require any energy.

The amino acids are combined with a peptide bond (-CONH-), where carboxyl groups of some amino acid is bound to amino groups of other amino acid molecules to produce some dipeptide by releasing water molecules (Araujo, 2010). The dipeptide still has amino acid groups and free carboxyls so as to be able to react with other dipeptides and finally build some protein molecules.

CONCLUSION

The best culture is technical Guillard; the conclusion is based on the growth of Chaetoceros gracilis, which reached its peak in technical Guillard culture media. The stationary phase duration of the growth influences the nutrient absorption in the culture media; the conclusion is based on the analysis of fatty acid profile and essential amino acid Chaetoceros gracilis, which reached its peak in technical Guillard culture media.

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Index of Images

  • Fig. 1 The growth of C. gracilis in double Walne and technical Guillard culture media

    Fig. 1 The growth of C. gracilis in double Walne and technical Guillard culture media

  • Table 1 Double Walne and technical Guillard culture media which were utilized to culture Chaetoceros gracilis

    Table 1 Double Walne and technical Guillard culture media which were utilized to culture Chaetoceros gracilis

  • Table 2 Quality of culture media water of the research

    Table 2 Quality of culture media water of the research

  • Table 3 The growth phase of C. gracilis in technical Guillard and double Walne culture media

    Table 3 The growth phase of C. gracilis in technical Guillard and double Walne culture media

  • Table 4 The fatty acid profile of Chaetoceros gracilis in double Walne dan technical Guillard culture media

    Table 4 The fatty acid profile of Chaetoceros gracilis in double Walne dan technical Guillard culture media

  • Table 5 Essential amino acid profile Chaetoceros gracilis in culture media double Walne dan technical Guillard<BR>

    Table 5 Essential amino acid profile Chaetoceros gracilis in culture media double Walne dan technical Guillard

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