KR20190061268A - Novel microorganism bacillus subtilis cjbb-121 or additives for fish feeds containing its growth medium - Google Patents

Abstract

The present invention relates to a Bacillus subtilis CJbb-121 strain or a feed additive for fish containing the same. The Bacillus subtilis CJbb-121 strain has an excellent decomposing activity of acid treated Gelidium elegans residue, and a culturing solution obtained by culturing Bacillus subtilis CJbb-121 strain in a microorganism medium containing the acid treated Gelidium elegans residue improves growth of fish including halibut. Accordingly, the strain of the present invention and a culture solution thereof use, in an eco-friendly way, Gelidium elegans residue which has been classified as waste after manufacturing agar, and enable mass production of fish at a low cost.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel microbial Bacillus subtilis CJbb-121 strain or a feed additive for a fish including a culture thereof. ≪ RTI ID = 0.0 >

The present invention relates to a novel microorganism Bacillus subtilis CJbb-121 strain showing a decomposing activity of mugwort, or a fish food comprising a cultured medium obtained by inoculating the new microorganism with an acid-treated mugwort residue and molasses mixture ≪ / RTI >

Currently, in fish farms, problems such as increased disease caused by dense farming, nutritional imbalance and other factors are causing serious problems such as fish growth and mortality.

Additives are added to the feed to solve the above problems. Commonly used feed additives include antibiotics, vitamins, amino acid feeders, mineral feeders, enzymes, and hormones.

However, the above-mentioned feed additives may cause drug abuse due to an increase in tolerance to chemicals of cultured fish in the case of excessive feeding, so that the harmful substances accumulate in the cultured fish, The fact that indirect consumption of harmful substances has a problem in the safety of marine products. In order to solve this problem, studies on various eco-friendly fish feed compositions have been carried out. However, studies on feed additive compositions, which are mostly made of expensive raw materials for natural materials and can be produced at low cost, Step.

Gelidium amansii LAMOUROUX is a marine algae belonging to the genus Gracilaria, which produces gametophytes as a sexually reproducing gonorrhea. It is washed in fresh water and it is washed in fresh water to remove the residue (byproduct). The inventors of the present invention conducted a study on eco-friendly fish feed additive that can be supplied to a large quantity of fish, and found that a new strain of microorganism Bacillus subtilis was produced by treating wastes treated as waste and decomposing residual wastes by- It has been confirmed that the microbial degraded by-products of the present invention have an excellent effect and that the microorganism-degraded by-products of the present invention have an effect of promoting the growth of fishes.

Korean Patent No. 10-1480028 (entitled " New microorganism Bacillus subtilis SJ-30 " and feed additives for fishes containing the same) Korean Patent No. 10-057771 (a composition for adding a fish feed containing Bacillus polyfermentus, Bacillus licheniformis and Saccharomyces cerevisiae) Korean Patent No. 10-860111 (the name of the invention: new microbial strain Truce Tokyatium SPK. Kasez-1, Bacillus polyfermenticus kejese-2 and feed additives including these and Bacillus licheniformis And feed) Korean Patent No. 10-1316086 (a new microorganism having agar and carrageenan resolution)

The present invention is to provide a feed additive for fish comprising a novel microorganism Bacillus subtilis CJbb-121 strain exhibiting decomposition activity of Agaricus edulis or agar, or a by-product of fermentation by fermentation using the new microorganism.

The present invention relates to a Bacillus subtilis CJbb-121 strain ( Bacillus subtilis CJbb-121, Accession No. KCTC18622P), which is characterized by exhibiting decomposition activity of an acid-treated mugwort residue.

The acid-treated starch residue may be an acid-treated product of the remaining starch residue body after removing the liquid from the hot water extract of the starch. In addition, the residue of the acid-treated mugwort can be adjusted to pH 7.0 ± 0.2 after mixing the mugwort body with the sulfuric acid solution at 12.5 to 62.5 g / l.

The present invention also relates to a feed additive for fish containing a culture obtained by culturing a strain of Bacillus subtilis CJbb-121 in a microorganism medium containing an acid-treated starch residue and a carbon source.

The carbon source may be selected from molasses or dextrin.

It is preferable that the carbon source is added to the microorganism medium at 10 to 30 g / l.

Hereinafter, the present invention will be described in detail.

The acid-treated starch residue which can be used in the present invention may be an acid-treated product of the remaining mugwort residue body after removing the liquid from hot water extract of Mugwort-mugwort. More preferably, the acid-treated mugwort residue is adjusted to pH 7.0 ± 0.2 . When water is extracted from the mugwort, the water soluble substance such as agar is extracted. The remaining mugwort body includes fibrils forming the shape of mugwort, and some of the agar may remain. However, the strain of the present invention decomposes the agar And the agar is not used for the growth and growth of the strain. In the present invention, the mugwort body can also be referred to as a mulberry, a residue, a by-product, etc. after removing the liquid phase after extraction.

The acid-treated groundwood residue may be the one having the pH adjusted to 7.0 ± 0.2 after mixing the watercress body with the sulfuric acid solution at 12.5 to 62.5 g / l. At this time, if the body of the mugwort is added at a content of less than 12.5 g / L or added at a content of more than 62.5 g / L, the strain of the present invention may not be well cultivated so that the mugwort body may not be easily decomposed. It may not be appropriate.

As the sulfuric acid solution used for the acid treatment, a 0.5 to 3% (w / v) sulfuric acid aqueous solution is preferably used. When the concentration of the sulfuric acid solution is less than 0.5% (w / v)%, the hydrolysis of the watercress body may not be performed well, so that the strain of the present invention is difficult to further decompose the watercress body, And when the concentration of the sulfuric acid solution exceeds 3% (w / v)%, the acidity may increase and the growth of the strain may be inhibited, so that the strain may be difficult to decompose.

The Bacillus subtilis CJbb-121 strain of the present invention is characterized by comprising the nucleotide sequence of SEQ ID NO: 1.

The Bacillus subtilis CJbb-121 strain can be cultured in a conventional microorganism medium, and the culture medium of the Bacillus subtilis CJbb-121 strain of the present invention can be obtained by inoculating a strain of Bacillus subtilis CJbb-121 into a conventional microorganism medium It may be cultured.

However, in order to produce a fish feed composition that is produced in order to efficiently carry out mass culture of fish in a less cost effective manner, the culture of Bacillus subtilis CJbb-121 strain is inoculated into a culture composition containing acid- It is better to use.

The medium composition may contain molasses or dextrin as a carbon source, and molasses is preferably used. Molasses refers to a concentrated sugar solution after the sugar is recovered from a mixture of sugar crystals and mother liquor obtained by crystallization of the sugar solution when preparing the sugar beet, tablets and bitter sugars (bagasse).

The carbon source is preferably added to the medium composition in an amount of 10 to 30 g / l. When the carbon source is added to the culture medium at a concentration of less than 10 g / L, the growth of Bacillus subtilis CJbb-121 strain may not be good. If the carbon source is added in an amount exceeding 30 g / L, the Bacillus subtilis The quality of the CJbb-121 culture medium may deteriorate and the growth of the fish may not be good.

The culture time after inoculation of the Bacillus subtilis CJbb-121 strain of the present invention into the culture composition is preferably 12 to 48 hours, more preferably 18 to 30 hours. The culture temperature of the strain of Bacillus subtilis CJbb-121 is preferably 30 to 40 占 폚. Even when the incubation time and the incubation temperature are not maintained, the strain of Bacillus subtilis CJbb-121 can not grow well or overgrowth.

In the present invention, the culture broth of Bacillus subtilis CJbb-121 strain used in the feed additives for fish can be used as it is, but only the remaining liquid after removing the cells can be used, and the culture can be concentrated or dried It is possible.

The culture medium of the Bacillus subtilis CJbb-121 strain of the present invention can be applied as a feed additive, but it is preferably used for raising flounder or rockfish.

In addition, the feed additives for fish of the present invention may include various excipients used in general feed additives for fish.

The present invention relates to a feed additive for Bacillus subtilis CJbb-121 strain or a fish comprising the same.

The Bacillus subtilis CJbb-121 strain is excellent in decomposition activity of the acid-treated mugwort residue and the culture solution obtained by culturing the Bacillus subtilis CJbb-121 strain in the microorganism medium containing the acid-treated mugwort residue is the fish such as the flounder The effect of the present invention is enhanced. Thus, the strain of the present invention and the culture medium of the present invention can be eco-friendlyly used as a residue of Ugukgasari which has been left as a waste after KEPCO manufacturing, and mass production of fish at low cost has become possible.

Korean Patent No. 10-1480028 discloses a technique for preparing a feed composition based on a strain of the genus Bacillus, but it has not yet been confirmed as a prior art that a feed composition for fish can be prepared using a by-product of the present invention . Although the strain of Bacillus sp. Having the ability of resolving agar is disclosed in Korean Patent No. 10-1316086, the strain of the present invention has the ability of resolving the residual agar residue after agar production, which is not an agar resolving ability, Technology.

FIG. 1 is a phylogenetic tree showing the result of phylogenic tree of Bacillus subtilis CJbb-121 strain.
FIG. 2 is a graph showing the number of viable cells of the culture medium of Bacillus subtilis CJbb-121 according to the amount of acid-treated starch residue added in the culture medium composition.
FIG. 3 is a graph showing the number of viable cells of the culture medium of Bacillus subtilis CJbb-121 according to the type of carbon source in the culture medium composition.
4 is a graph showing the number of viable cells of the strain Bacillus subtilis CJbb-121 according to the molasses concentration in the culture medium.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

≪ Example 1: Isolation and growth of strain &

Example 1-1. Screening and isolation of strain

Extracts of agar from mulberry leaves are mostly discarded. Although the residue itself is composed of carbohydrates, it is not easy to use the residue as it is. Therefore, in the present invention, an application method which can decompose the residue of microcrystalline cellulose into microorganisms and use it as a feed additive for fish has been sought.

The microorganisms suitable for decomposing residues were collected from salted and marine soil samples collected from 12 different areas of Jeju Island. The sample was diluted 10 ³ times with sterilized physiological saline and cultured in a culture medium (5 g of yeast extract, 5 g of NaCl, ₂ g of K ₂ HPO ₄ , ₁ g of MgSO ₄ .7H ₂ O, 1 g of KCl, 0.5 g of CaCl ₂ and 15 g of agar) A total of 136 single colonies were obtained by culturing at 30 ° C for 3 days. The resulting colonies were sufficiently cultured for 24 hours and then inoculated into a liquid culture medium (5 g of yeast extract, 5 g of NaCl, ₂ g of K ₂ HPO ₄ , ₁ g of MgSO ₄ .7H ₂ O, 1 g of KCl and 0.5 g of CaCl ₂ ) Respectively.

The agar was extracted from the 136 strains and the resolving power for the remaining by-products was confirmed. First, for the production of agar, 10-fold weight of water was added to watercress to obtain a hot-water extract, and the extract was removed to obtain a residue (body, mackerel) of the remaining watercress. In the preliminary experiment, it was confirmed that the decomposition efficiency of the residue of the mugwort residue was high when the acid residue was decomposed by microorganism. Therefore, after adding 4 g of the residue of the mugwort residue and 160 ml of 1% sulfuric acid solution, the mixture was pulverized for 2 minutes using a blender, And the pH was adjusted to 7.0 ± 0.2 using 5N NaOH.

1% of the culture broth of the pre-cultured separated microorganism was inoculated into the liquid phase of sulfuric acid-treated mugwort residue (the body of sulfuric acid-treated mugwort) and cultured at 30 ° C for 24 hours at 150 rpm. Then, the culture medium (yeast extract 5g, , K2HPO4 (2 g), MgSO4 7H2O (1 g), KCl (1 g), CaCl2 (0.5 g) and agar (15 g) were serial diluted and incubated at 30 ° C for 2 days. The decomposition activity of Sulfuric acid-treated Staphylococcus aureus is 1.0 x 10 ⁴ cfu / ml - 1.0 x 10 cfu / ml based on the number of colonies identified after culturing, + + 1.0 x 10 ⁵ cfu / + Was less than 1.0 × 10 ⁶ cfu / ml, and +++ was 1.0 × 10 ⁶ cfu / ml or greater.

Of the total 136 strains, 103 strains showed resolving activity against the sulfate residue treated with '+' sulfuric acid. Among them, the inventors of the present invention found that a strain capable of decomposing the residues of Staphylococcus aureus and using the degraded Staphylococcus aureus as feed additives Were 9 species. Bacillus subtilis (++), Bacillus sp., Isolated from fish culture, Two species (all ++), Bacillus sp ., Isolated from anchovy salted fish. Is-2 1 species (++), soil separation Bacillus subtilis CJbb-121 1 species (+++), citrus peel separation Bacillus sp. (All ++). Among them, Bacillus subtilis CJbb-121 (+++) isolated from soil was finally selected as a strain of the present invention in consideration of the decomposition of sulfuric acid decomposing residue.

Examples 1-2. Confirm the secretion of agarase

In order to compare the activity of the screening strains with those of strains mainly digesting agar of the seaweed, the agarase secretion ability was first confirmed by the following method. The culture broth of this strain was plated on a new solid culture medium (5 g of yeast extract, 5 g of NaCl, ₂ g of K ₂ HPO ₄ , ₁ g of MgSO ₄ .7H ₂ O, 1 g of KCl, 0.5 g of CaCl ₂ and 15 g of agar) And agar degradation activity was confirmed by agar digestion activity. As a result, all of these strains did not digest agar and showed no special agarase secretion function.

Examples 1-2. 16S rRNA sequencing

For 16S ribosomal RNA analysis, Chromosomal DNA was isolated using Genomic DNA Extraction kit (Bioneer, Korea) and 16S rDNA was amplified using Bacterial 16S rDNA universal primer. Each primer of the synthesized oligonucleotides consisted of forward primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3 'reverse primer (1492R): 5'-GGTTACCTTGTTACGACTT-3', and each 0.5 μM primer, 200 μM deoxynucleoside triphosphate, Taq PCR was performed using 3 μl of DNA polymerase (Bioneer, Korea). The PCR reaction conditions were predenaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 45 seconds, annealing at 50 ° C for 45 seconds, extension at 72 ° C for 45 seconds, and extension at 72 ° C for 5 minutes. The amplified PCR product was electrophoresed with ethidium bromide and confirmed in 1% agarose gel (Promega Co., USA) gel. Afterwards, the remaining primers, nucleotides, polymerase and salts were removed from the PCR product using the Accuprep ™ PCR purification kit (Bioneer, Korea) and the DNA was eluted with 30 μl of elution buffer (10 mM Tris-Cl, pH 8.5) . The nucleotide sequence of the PCR product was analyzed using ABI prism ™ Bigdye ™ terminator cycle sequencing Ready reaction kit V.3.1 (Fluorescent dye terminators method) and ABI 3730XL capillary DNA sequencer. Homology analysis of DNA sequences was performed using the BLAST online program (http://ncbi.nlm.nih.gov/blast). The nucleotide sequences were then sequenced using the ClustalW software and a phylogenetic tree was constructed using the MEGA3 program (Ver 3.0).

The 1399 bp sequence (SEQ ID NO: 1) obtained by 16S ribosomal RNA PCR amplification of Bacillus subtilis CJbb-121 was analyzed using NCAI (National Center Biotechnology Information) Basic Local Alignment Search Tool (BLAST). As expected, Bacillus subtilis 99% homology. Bacillus subtilis, Bacillus velzensis, and Bacillus amyloliquefaciens ) 16S rRNA sequences and type strain 16S rRNA sequences of Bacillus spp . (Fig. 1). As a result, the isolated Bacillus subtilus CJbb-121 was found to be a novel Bacillus subtilis strain 99% similar to the above strain.

SEQ ID NO: 1:

≪ Example 2: Optimization process of microorganism culture composition I - Determination of addition amount of Leucocephalus residue and addition amount of sulfuric acid >

In order to determine the amount of Leucocephalus residue added to the culture medium for the cultivation of the strain Bacillus subtilis CJbb-121, the total volume of Leucillus japonicus was changed to 0 g, 2 g, 4 g, 6 g, 8 g and 10 g, A 1% (w / v) sulfuric acid solution was added and homogenized by grinding with a grinder to adjust the pH to 7.0 ± 0.2 and sterilized. The sterilized medium was inoculated with 1% by weight of Bacillus subtilis CJbb-121 (pre-culture solution) and cultured. Extracts were extracted from water extracts twice for agar production, and then only the remaining extracts were collected and used.

The number of viable cells in the culture was confirmed under the conditions of Example 2. As a result, the highest viable cell count was confirmed at 4 g / 160 ml (25 g / l) as shown in FIG. 2, but under the condition of 2 g / 160 ml to 10 g / g / ℓ), the cultivation of Bacillus subtilis CJbb-121 strain is smooth.

The concentration of sulfuric acid solution was 0.5 ~ 3 (w / v)%, and it was confirmed that the number of viable cells was higher than that of other solutions.

≪ Example 3: Optimization process of microorganism culture composition II - Identification of carbon source type and confirmation of addition amount >

For the preparation of agar, the extracts were extracted twice with hot water. The remaining 4 g of the extract was mixed with 160 ml of a 1% (w / v) solution of sulfuric acid and homogenized with a grinder to adjust the pH to 7.0 ± 0.2 with 5N NaOH. , Molasses, and dextrin as a carbon source were added at a concentration of 20 g / l and sterilized. The sterilized medium was inoculated with 1% by weight of a strain of Bacillus subtilis CJbb-121 (pre-culture solution), and the number of viable cells in the culture was confirmed under the conditions of Example 2. [ The results are shown in FIG. 3, in which cane molasses was found to have the highest viable count, and addition of dextrin also enhanced the growth of Bacillus subtilis CJbb-121 strain.

Next, the number of viable cells was determined according to the concentration of the molasses in the same manner. Referring to FIG. 4, when the molasses concentration was 10 to 30 g / L, the number of viable cells was high, and 25 g / L was the best.

In addition, the addition of a nitrogen source (yeast extract, meat peptone, soy peptone) and an enzyme (cellulase) as a source of the medium composition was carried out. However, the growth promoting efficiency of the strain and the degrading activity of the starch- And the growth promotion effect of the fish was not different from that of the addition of the carbon source alone. The addition of a nitrogen source or enzyme does not meet the intention of reducing the production cost for recycling and mass production of disused resources because the production cost of the feed composition for fish is greatly increased and does not meet the objective of growth / It was confirmed that the growth of the fish was promoted efficiently only by the addition of the carbon source. Therefore, only the carbon source was added to the medium composition used in the present invention.

≪ Example 4: Confirmation of effect of feed additive on fish growth >

25 g of the residue of the agar remaining after the preparation of agar and 10 l of 1% (w / v) sulfuric acid were mixed and pulverized with a mixer evenly. After grinding, the pH was adjusted to pH 7.0 +/- 0.2 with 5N NaOH and sterilized by addition of 250 g molasses. After that, Bacillus subtilis CJbb-121 strain was inoculated and cultured at 37 캜 for 24 hours to prepare a feed additive composition for fish.

The culture was diluted to a concentration of 1.0 x 10 ⁶ cfu / g of Bacillus subtilis CJbb-121 strain, 1 g and 3 g were sprayed, and the flounder was divided into 30 legs (Total length) and body weight of the flounder were measured. ** The concentration of Bacillus subtilis CJbb-121 strain in the culture medium was found to be most suitable for the weight gain of flounder fed from 5.0 × 10 ⁵ cfu / g to 1.0 × 10 ⁷ cfu / g through preliminary experiments.

Experimental groups were divided into two groups: a control group in which the culture was not added and only feeds were fed, an experimental group (experimental group 1) in which the culture medium contained 1 g of feed per 100 g of feed, an experimental group in which the culture medium contained 3 g per 100 g of feed 2) for a total of 16 weeks. Then, the body size and body weight of the flounder were measured.

first final Number of flounder Length (cm) Weight (g) Number of flounder Length (cm) Weight (g) Control group 30 23.5 133.4 20 28.7 218.1 Experiment 1 30 23.5 132.8 26 28.9 224.9 Experiment 2 30 23.6 132.9 28 29.5 241.3

As shown in Table 1, it can be seen that the weight of the flounder greatly increased in the flounder fed with the culture solution (feed additive of the present invention) after 16 weeks.

In addition to the above results, the addition of 5 g of the reference culture medium of 100 g of feed was more remarkable for the increase in body weight and body weight, and sufficient body weight and weight gain effect was confirmed even in the case of supplying 5 g, I did not confirm it.

[Agency]

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC18622P

Funding date: 20170927

<110> OH, MYUNG CHEOL          JEJUAGAR          Lemna Science <120> NOVEL MICROORGANISM BACILLUS SUBTILISIS CJBB-121 OR ADDITIVES FOR          FISH FEEDS CONTAINING ITS GROWTH MEDIUM <130> 0326 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1405 <212> DNA <213> BACILLUS SUBTILIS <400> 1 atgcagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60 acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120 atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc acttacagat 180 ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag 240 ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300 ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360 gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac cgttcgaata 420 gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480 ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540 tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600 aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660 tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720 gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780 agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840 gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960 tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080 ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140 tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200 caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260 gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380 cccgaagtcg gtgaggtaac ctttt 1405

Claims (6)

Bacillus subtilis CJbb-121 strain ( Bacillus subtilis CJbb-121, Accession No. KCTC18622P), which exhibits decomposition activity of an acid-treated mugwort residue. The method according to claim 1,
( Bacillus subtilis CJbb-121, Accession No. KCTC18622P), which is an acid-treated product of the mugwort residue body obtained by removing the liquid phase from hot water extract of the mugwort, and the acid treated mugwort residue. 3. The method of claim 2,
The Bacillus subtilis CJbb-121 strain ( Bacillus subtilis CJbb-121), which is obtained by mixing the sulfuric acid solution with 2 g: 16 ml to 10 g: 16 ml and adjusting the pH to 7.0 +/- 0.2, 121, accession number KCTC18622P). A feed additive for fish characterized by containing a culture obtained by culturing the strain of claim 1 in a microorganism medium containing an acid-treated starch residue and a carbon source. 5. The method of claim 4,
Wherein the carbon source is selected from molasses or dextrin. A feed composition for fish culture comprising the feed additive of claim 1.

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