TW201902363A - Use a mixture of three strains of lactic acid bacteria to improve the dissolved oxygen in the aquaculture pond - Google Patents

Abstract

This invention discloses that a mixture of Lactobacillus plantarum BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus fermentum LF26 can be used in improving dissolved oxygen in an aquaculture pond, wherein Lactobacillus fermentum LF26 is deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) under accession number BCRC 910752.

Description

Use a mixture of three lactic acid bacteria strains to improve dissolved oxygen in aquaculture ponds

The invention relates to a method for increasing the dissolved oxygen content of an aquaculture pond, which comprises: administering to the aquaculture pond a bacterium containing Lactobacillus embryos BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus LF26 Among them, Lactobacillus fermentum LF26 is deposited at the Biological Resources Preservation and Research Center of the Institute of Food Industry Development under the registration number BCRC 910752.

In aquaculture ponds, the continuous accumulation of wreckage and excrement of aquaculture animals and the remaining feed will cause the amount of dissolved oxygen to decrease, which will not only promote anaerobic decomposition and produce toxic substances, but also promote the growth of pathogenic bacteria. The speed of feeding, digestion, metabolism, and growth of aquaculture animals, as well as their activity and resistance, may also be affected by low dissolved oxygen levels, and severe cases may even die. For example, in terms of fish and shrimp farming, the amount of dissolved oxygen must be 4-8 mg / L and 6-8 mg / L, respectively, to meet the growth of fish and shrimp.

The current methods used to improve the dissolved oxygen content of aquaculture ponds include the following two methods: (1) mechanical aeration: using the mechanical action of an aerator to promote water contact with the air and promote convection of the water body; and (2) Chemical Oxygenation: Chemical oxygenation agents (for example, sodium peroxide, calcium peroxide, etc.) are added to water and release oxygen by chemical reaction. However, mechanical aeration requires a large number of aerators and energy to significantly increase the cost. As for chemical aeration, metal ions and pH values in water bodies will increase, which may adversely affect the health of aquaculture animals, and may even It will cause toxic substances to remain in the body and be absorbed into the body. Therefore, the development of low-cost aeration technology without safety concerns has become an important research and development issue.

Lactic acid bacteria (LAB) are a group of Gram-positive bacteria that can ferment sugars and use lactic acid as the main metabolite. They are commonly found in dairy products, pickles, and intestinal mucosa of humans or animals. Lactobacillus has been widely accepted morphological and physiological characteristics include: (1) the shape is spherical (cocci) or rod (rod); (2) lack of cytochrome and catalase (3) does not form Endophytic spores; and (4) non-motile.

Lactic acid bacteria are generally recognized as safe (GRAS) and are familiar and widely used probiotics. Lactic acid bacteria have been found to have the effects of inhibiting the growth of gastrointestinal pathogens, alleviating lactose intolerance, immunoregulation, anti-cancer, and lowering blood pressure. It can be used as a probiotic lactic acid bacteria used in many species, such as Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), Pediococcus (Pediococcus), Streptococcus (Streptococcus) and Enterococcus (Enterococcus) and so on.

Studies have shown that some lactic acid bacteria strains have the effect of reducing nitrate concentration in aquaculture ponds. For example, in Song Rong et al. (2013), Freshwater Fisheries, 43: 1-8, Song Rong et al. Found that Lactobacillus casei L821a is capable of undergoing intracellular enzymolysis and metabolite lactic acid Direct and indirect action to remove nitrate in aquaculture waters. However, as far as the applicant is aware, no literature or pre-patent case has so far revealed that the lactic acid bacteria strain has the effect of improving the dissolved oxygen amount in the aquaculture pond.

In previous research, the applicant found that a bacterium containing Lactobacillus plantarum BCRC 910435 (also known as Lactobacillus plantarum LP28 or Lactobacillus plantarum LP28), Pediococcus pentosaceus BCRC 910480 (also known as It is a mixture of Pediococcus pentoseus PP4012 or Pediococcus pentoseus PP49), Lactobacillus fermentum , Lactobacillus rhamnosus , Lactobacillus paracasei , and Bacillus subtilis ( Trade name Shui Yiwang) can improve the gastrointestinal function of fish, enhance the immunity of fish and shrimp, feed exchange rate and growth rate. After further research, the applicant unexpectedly discovered that a mixture containing Lactobacillus germium BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus fermentum LF26 can improve the dissolved oxygen content of the aquaculture pond.

Summary of invention

Therefore, the present invention provides a method for increasing the dissolved oxygen content of an aquaculture pond, which comprises: administering to the aquaculture pond a bacterium containing Lactobacillus embryos BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus LF26 Among them, Lactobacillus fermentum LF26 is deposited at the Biological Resources Preservation and Research Center of the Institute of Food Industry Development under the registration number BCRC 910752.

The above and other objects, features and advantages of the present invention will become apparent after referring to the following detailed description and preferred embodiments and accompanying drawings.

Detailed description of the invention

For the purpose of this specification, it will be clearly understood that the word "comprising" means "including but not limited to" and the word "comprises" has a corresponding meaning.

It should be understood that if any previous case publication is cited here, the previous case publication does not constitute an admission that, in Taiwan or any other country, the former case publication forms a common generality in the art. Part of the knowledge.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Of course, the invention is in no way limited by the methods and materials described.

In order to develop a low-cost aquaculture pond aeration technology without safety concerns, the applicant's research results found that a mixture containing Lactobacillus germophilus BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus fermentum LF26 has this aspect Industrial application potential. Therefore, the present invention provides a method for increasing the dissolved oxygen content of an aquaculture pond, which comprises: administering to the aquaculture pond a bacterium containing Lactobacillus embryos BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus LF26 Among them, Lactobacillus fermentum LF26 is deposited at the Biological Resources Preservation and Research Center of the Institute of Food Industry Development under the registration number BCRC 910752.

According to the invention, the aquaculture pond may be a shrimp pond. Preferably, the shrimp is selected from the group consisting of: Litopenaeus vannamei (also known as Pacific white shrimp), freshwater prawn ( Macrobrachium rosenbergii ) (also known as Macrobrachium rosenbergii), Penaeus monodon (also known as Penaeus monodon), and combinations thereof. In a preferred embodiment of the present invention, the shrimp is P. vannamei.

According to the present invention, the shrimp breeding pond may have a stocking density ranging from 50 / m ^ 2 to 250 / m ^ 2. In a preferred embodiment of the present invention, the shrimp breeding pond has a breeding density of about 91 birds per square meter.

According to the invention, the aquaculture pond may be a fish pond. Preferably, the fish is selected from the group consisting of: Epinephelus fuscoguttatus (also known as dragon tiger grouper), Epinephelus lanceolatus (also known as gentian grouper) , Spotted grouper ( Epinephelus coioides ) (also known as blue spot), and combinations thereof. In a preferred embodiment of the present invention, the fish is a brown spot grouper.

According to the present invention, the fish breeding pond may have a breeding density ranging from 5 animals / square meter to 12 animals / square meter. In a preferred embodiment of the present invention, the fish breeding pond has a breeding density of about 6 animals per square meter.

According to the present invention, among the mixture, Lactobacillus brevis BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus fermentum LF26 may fall in a range from 1: 1: 1 (w / w / w) to 1: 2: 1 (w / w / w). In a preferred embodiment of the present invention, the Lactobacillus brevis BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus fermentum LF26 are in a ratio of 1: 2: 1 (w / w / w).

According to the invention, the mixture may have a bacterial concentration ranging from 10 ⁵ CFU / g to 10 ⁹ CFU / g. In a preferred embodiment of the present invention, the mixture has a bacterial concentration ranging from 10 ⁶ CFU / g to 10 ⁹ CFU / g.

According to the present invention, the mixture may further include a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of a solvent, a buffer, an emulsifier, and a suspending agent. agent), decomposer, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, Gelling agents, wetting agents, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents fall within the professionalism and routine skills of those familiar with the technology.

According to the invention, the mixture can be added to aquaculture feed using a standard technique known to those skilled in the art, for example, it can be added directly to aquaculture feed, or it can be used In producing one or more intermediate compositions [such as feed additives or premixes], the intermediate compositions are then added to the aquaculture feed. In a preferred embodiment of the present invention, the mixture is administered to the aquaculture pond after being mixed with an aquaculture feed.

According to the invention, the mixture and aquaculture feed can be mixed in a range of ratios ranging from 1: 100 (w / w) to 1: 999 (w / w). In a preferred embodiment of the present invention, the mixture is mixed with the aquaculture feed at a ratio of 1: 999 (w / w).

Detailed description of the preferred embodiment

The present invention will be further described with reference to the following examples, but it should be understood that these examples are for illustrative purposes only and should not be construed as limitations on the implementation of the present invention. Examples General experimental materials: 1. Lactobacillus plantarum BCRC 910435 and Pediococcus pentosaceus BCRC 910480:

Lactobacillus brevis BCRC 910435 (also known as Lactobacillus lactis LP28 or Lactobacillus plantarum LP28) and Pediococcus pentosus BCRC 910480 (also known as Pediococcus pentosus PP4012 or Pediococcus pentosus PP49) were deposited at the Food Industry Research and Development Institute (FIRDI) 's Biosource Collection (Biosource Collection) on July 14, 2009 and July 22, 2010. and Research Center, BCRC) (No. 331 Food Road, Hsinchu City, Taiwan), and it can be freely assigned.

In addition, these two strains were also deposited under the Budapest Treaty on October 19, 2009 and September 9, 2011 under the deposit numbers CGMCC No. 3346 and CGMCC No. 5235, respectively. In China General Microbiological Culture Collection Center (CGMCC), and can be freely assigned. 2. Lactobacillus fermentum LF26:

The applicant previously used dairy products as a sample source and used Difco Lactobacilli MRS agar (Difco Lactobacilli MRS Agar) to isolate and screen Lactobacillus to obtain 1 Lactobacillus isolate LF26. The Lactobacillus isolate LF26 was found through preliminary tests. It is a Gram-positive bacterium, does not have enzymes, and can grow under anaerobic conditions. In addition, the Lactobacillus isolate LF26 was found through acid resistance, salt tolerance, and bile salt tolerance tests, and it can survive in pH 2.0, 5% NaCl, and 0.3% bovine bile environments.

This Lactobacillus isolate LF26 was subjected to 16S rDNA sequence analysis using a set of universal primer pairs F1 and R1 with the nucleotide sequences shown below (commissioned by Mingxin Biotechnology Co., Ltd.) and used NCBI website provides Gene Blast software for comparison: F1 primer 5'-agagtttgatcmtggctcag-3 '(sequence identification number: 1) R1 primer 5'-cggttaccttgttacgactt-3' (sequence identification number: 2)

The comparison results revealed that the 16S rDNA sequence (sequence identification number: 3) of the part of the Lactobacillus isolate LF26 had the highest sequence similarity between the 16S rDNA sequence of Lactobacillus fermentum and the 16S rDNA sequence.

This Lactobacillus isolate LF26 was subjected to pheS gene sequence analysis using a set of universal primer pairs F2 and R2 with the nucleotide sequences shown below (commissioned by Mingxin Biotechnology Co., Ltd.), and provided by Gene Blast software for comparison: F2 primer 5'-cayccngchcgygayatgc-3 '(sequence identification number: 4) R2 primer 5'- ccwarvccraargcaaarcc-3' (sequence identification number: 5)

As a result of comparison, it was found that the pheS gene (sequence identification number: 6) of the LF26 part of the Lactobacillus isolate has the highest sequence similarity with the pheS gene sequence of Lactobacillus fermentum.

This lactobacillus isolate LF26 was further analyzed for a sugar fermentation pattern using an API 50 CHL identification system (bioMérieux). The obtained identification results are shown in Table 1 below. Table 1. Results of sugar fermentation analysis of Lactobacillus isolate LF26

Based on the results of the above experiments, the Lactobacillus isolate LF26 was identified as Lactobacillus fermentum. The Lactobacillus fermentum LF26 was deposited at the Biological Resource Conservation and Research Center of the Food Industry Development Institute of Taiwan on November 3, 2016 under the deposit number BCRC 910752. 3. Experimental animals:

Litopenaeus vannamei (7 days old, weighing about 0.0034 g) and Epinephelus fuscoguttatus (244 days old, weighing 324 g) used in the following examples were purchased from The shrimp fry farm in Kaohsiung Forest Park (known as the Linyuan Black Shell Park) and the grouper fish farm in Yongan District, Kaohsiung City. All experimental animals were kept in open-air aquaculture ponds, and feed was adequately supplied. Example 1. Preparation of a mixture of the present invention (a mixture of three lactic acid bacteria strains) containing lactic acid bacteria 3

First, Lactobacillus brevis BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus fermentum LF26 were respectively inoculated into 4.5 L of the inoculum culture medium shown in Table 2 below with a 3% (v / v) inoculation amount, and The culture was carried out at 37 ° C for 18 hours. Then, all the cultures were collected and inoculated into 150 L of inoculum culture medium, respectively, and then cultured at 37 ° C for 16 hours. Thereafter, the cultures were centrifuged at 12,000 rpm at 4 ° C for 30 minutes, and then the supernatant liquid was removed, and the pellets were washed with an appropriate amount of physiological saline. After the plate count medium was used to count the number of bacteria, freeze-drying treatment was performed to obtain freeze-dried powder of each strain (each had a bacterial concentration of 10 ¹¹ CFU / g). Table 2. Inoculum formulations

Thereafter, the freeze-dried bacterial powder of each strain and maltodextrin as a carrier were mixed with a formulation as shown in Table 3 below, thereby obtaining a mixture containing three lactic acid bacteria strains of the present invention (having 10 ⁹ CFU / g). Table 3. Formulation of the mixture of the present invention containing 3 strains of lactic acid bacteria Example 2. Experimental method for evaluating the effectiveness of a mixture containing 3 lactic acid bacteria strains of the present invention in improving the dissolved oxygen content of shrimp aquaculture ponds :

First, an aquaculture pond containing about 70,400 P. vannamei was used as an experimental group, and an aquaculture pond containing about 88,000 P. vannamei was used as a control group, and the stocking density of each group was about 91 / Square meter, and the water depth is about 100 cm. Next, feed was administered to the control group's aquaculture pond according to the time, feed type, and dose shown in Table 4 below. Table 4. Feed types and dosages administered at different times

The feed of the aquaculture pond in the experimental group was generally performed with reference to the control method of the control group, except that the feed was first added at a 0.1% (w / w) amount before being administered. The mixture containing 3 lactic acid bacteria strains of the present invention is added and mixed. Each group was fed with feed 3 times a day until the end of the 112th day after the start of feeding with lactic acid bacteria.

On the 49th and 112th days after the start of the lactic acid bacteria feed, an oxygen meter (brand: Lutron, model: DO-5510) was used to measure the dissolved oxygen content at 5 cm and 100 cm below the surface of each group. (mg / L) as the dissolved oxygen amount in the upper layer and dissolved oxygen amount in the lower layer, respectively.

The obtained experimental data is expressed by "mean ± standard error of the mean (SEM)". All data were analyzed by Student's t-test to assess differences between groups. If the obtained statistical comparison result is p <0.05, it means statistical significance. result:

The dissolved oxygen amount of each group is shown in Table 5 below. It can be seen from Table 5 that both the upper dissolved oxygen level and the lower dissolved oxygen amount of the experimental group will increase with time, while the upper dissolved oxygen amount and the lower layer dissolved oxygen amount of the control group will increase with time. Scenario of decline. In particular, on the 112th day, both the amount of dissolved oxygen in the upper layer and the amount of dissolved oxygen in the bottom layer of the experimental group were significantly larger than those in the control group. The results of this experiment show that the mixture containing the three lactic acid bacteria strains of the present invention can effectively increase the dissolved oxygen content of shrimp aquaculture ponds, and can be beneficial to the growth of shrimps. Table 5. Dissolved oxygen measured in each group (mg / L) **: When compared with the control group, p <0.01. ***: When compared with the control group, p <0.001. Example 3. Experimental method for evaluating the effectiveness of a mixture containing three lactic acid bacteria strains of the present invention in improving the dissolved oxygen content of fish aquaculture ponds

First, two aquaculture ponds each containing about 12,000 brown spotted grouper were used as the experimental group and the control group. The culture density of each group was about 6 per square meter, and the water depth was about 240 cm. Next, feed was administered to the aquaculture ponds of the control group at the time, feed type, and dose shown in Table 6 below. Table 6. Feed types and dosages administered at different times

The feed of the aquaculture pond in the experimental group was generally performed with reference to the control method of the control group, except that the feed was first added at a 0.1% (w / w) amount before being administered. The mixture containing 3 lactic acid bacteria strains of the present invention is added and mixed. Each group was fed with feed twice a day until the end of the 110th day after the start of feeding with lactic acid bacteria.

On the 82nd and 110th days after the start of the lactic acid bacteria feed, the oxygen levels (mg / L) at 5 cm, 84 cm, and 240 cm below the surface of each group were measured using an oxygen meter, respectively, as the upper layer dissolved oxygen amount. , The amount of dissolved oxygen in the middle layer and the amount of dissolved oxygen in the bottom layer. result:

The dissolved oxygen amount of each group is shown in Table 7 below. As can be seen from Table 7, during the entire experimental period, the amount of dissolved oxygen in the upper layer, the amount of dissolved oxygen in the middle layer, and the amount of dissolved oxygen in the bottom layer of the experimental group were significantly larger than those in the control group. The results of this experiment show that the mixture containing three lactic acid bacteria strains of the present invention can effectively increase the dissolved oxygen content of fish aquaculture ponds, and can be beneficial to the growth of fish. Table 7. Dissolved oxygen measured in each group (mg / L)

All patents and documents cited in this specification are incorporated by reference in their entirety. In case of conflict, the detailed explanation (including the definition) of this case will prevail.

Although the present invention has been described with reference to the specific examples described above, it will be apparent that many modifications and changes can be made without departing from the scope and spirit of the invention. Therefore, it is intended that the present invention is limited only by those indicated in the scope of patent application attached to the document.

TW Republic of China; Center for the Preservation and Research of Biological Resources of the Food Industry Development Institute (BCRC of FIRDI); 2016/11/3; BCRC 910752.

<110> Shenghe Biotechnology Co., Ltd. <120> Using a mixture containing three lactic acid bacteria strains to improve the dissolved oxygen content of aquaculture ponds <130> A mixture containing three lactic acid bacteria strains <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220><223> F1 primer for amplifying 16S rDNA <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 20 <212 ＞ DNA ＜ 213 ＞ Artificial sequence ＜ 220 ＞ ＜ 223 ＞ R1 primer for amplification of 16S rDNA ＜ 400 ＞ 2 cggttacctt gttacgactt 20 ＜ 210 ＞ 3 ＜ 211 ＞ 1538 ＜ 212 ＞ DNA ＜ 213 ＞ Lactobacillus fermentum <220 ＞ ＜ 221 ＞ misc_characteristics ＜ 222 ＞ (276) .. (276) ＜ 223 ＞ n stands for a, c, g or t ＜ 400 ＞ 3 gctcaggatg aacgccggcg gtgtggcctaa tacatgcaag tcgaacgcgt tggcccaatt 60 gattgatggt gcttgcacc cctggtcact gccgact gcctac gcc gaagcggggg acaacatttg gaaacagatg ctaataccgc 180 ataacaacgt tgttcgcatg aacaacgctt aaaagatggc ttctcgctat cacttctgga 240 tggacctgcg gtgcatta gc ttgttggtgg ggtaanggcc taccaaggcg atgatgcata 300 gccgagttga gagactgatc ggccacaatg ggactgagac acggcccata ctcctacggg 360 aggcagcagt agggaatctt ccacaatggg cgcaagcctg atggagcaac accgcgtgag 420 tgaagaaggg tttcggctcg taaagctctg ttgttaaaga agaacacgta tgagagtaac 480 tgttcatacg ttgacggtat ttaaccagaa agtcacggct aactacgtgc cagcagccgc 540 ggtaatacgt aggtggcaag cgttatccgg atttattggg cgtaaagaga gtgcaggcgg 600 ttttctaagt ctgatgtgaa agccttcggc ttaaccggag aagtgcatcg gaaactggat 660 aacttgagtg cagaagaggg tagtggaact ccatgtgtag cggtggaatg cgtagatata 720 tggaagaaca ccagtggcga aggcggctac ctggtctgca actgacgctg agactcgaaa 780 gcatgggtag cgaacaggat tagataccct ggtagtccat gccgtaaacg atgagtgcta 840 ggtgttggag ggtttccgcc cttcagtgcc ggagctaacg cattaagcac tccgcctggg 900 gagtacgacc gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag 960 catgtggttt aattcgaagc tacgcgaaga accttaccag gtcttgacat cttgcgccaa 1020 ccctagagat agggcgtttc cttcgggaac gcaatgacag gtggtgcatg gtcgtcgtca 1080 gctcgtgtcg tgagatgttg ggttaagtcc cgca acgagc gcaacccttg ttactagttg 1140 ccagcattaa gttgggcact ctagtgagac tgccggtgac aaaccggagg aaggtgggga 1200 cgacgtcaga tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacggta 1260 caacgagtcg cgaactcgcg agggcaagca aatctcttaa aaccgttctc agttcggact 1320 gcaggctgca actcgcctgc acgaagtcgg aatcgctagt aatcgcggat cagcatgccg 1380 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatgaga gtttgtaaca 1440 cccaaagtcg gtggggtaac cttttaggag ccagccgcct aaggtgggac agatgattag 1500 ggtgaagtcg taacaaggta gccgtaggag aacctgcg 1538 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <220><223> F2 primer for amplification of pheS gene <220><221> misc_ feature <222> (6). (6) <223> n stands for a, c, g or t <400> 4 cayccngchc gygayatgc 19 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220><223> Used for expansion R2 primer for pheS gene increase <400> 5 ccwarvccra argcaaarcc 20 <210> 6 <211> 431 <212> DNA <213> Lactobacillus fermentum <400> 6 gtgaccccgt ctgttttgat gcggac ccaa acgtcgccaa tgcaggcccg gatgctggaa 60 caacacgact tctccaaggg gccgttgaag atgatctcac cggggaaggt ttaccgccgc 120 gacaccgatg acgctaccca cagccaccaa ttccaccagg ttgaaggaat cgtggtcggt 180 gaacacgtca cgatggccga tttaaagggg accctagagg cggtggccca aaacctgttt 240 ggcgaccagc tcaaggtgcg tctgcgcccg agttacttcc cgttcacgga accgtccgtc 300 gaggccgaca tcacttgctt taattgcctg ggggccggtt gctcaatctg taagggaact 360 ggttggatcg aggtgttggg ggccggaatg gtgcacccaa acgtcttgaa gatgtctggc 420 gtcgacccgg a 431

Claims (12)

A method for increasing the amount of dissolved oxygen in an aquaculture pond, comprising: administering to the aquaculture pond a mixture comprising Lactobacillus sphaeroides BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus LF26, wherein: Lactobacillus fermentum LF26 is deposited at the Biological Resource Conservation and Research Center of the Food Industry Development Institute under the deposit number BCRC 910752. The method of claim 1, wherein the aquaculture pond is a shrimp breeding pond. The method of claim 2, wherein the shrimp is selected from the group consisting of white shrimp, freshwater long-leg prawn, grass prawn, and combinations thereof. The method of claim 3, wherein the shrimp is P. vannamei. The method of claim 1, wherein the aquaculture pond is a fish pond. The method of claim 5, wherein the fish is selected from the group consisting of brown spot grouper, saddle grouper, spot grouper, and combinations thereof. The method of claim 6, wherein the fish is a brown spot grouper. The method of claim 1, wherein in the mixture, Lactobacillus brevis BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus fermentum LF26 fall within a range of 1: 1 (w / w / w) to 1 : Ratio within 2: 1 (w / w / w). The method of claim 8, wherein in the mixture, the Lactobacillus brevis BCRC 910435, Pediococcus pentosaceus BCRC 910480, and Lactobacillus fermentum LF26 are in a ratio of 1: 2: 1 (w / w / w). The method of claim 1, wherein the mixture is administered to the aquaculture pond after being mixed with an aquaculture feed. The method of claim 1, wherein the mixture further comprises a pharmaceutically acceptable carrier. The method of claim 11, wherein the pharmaceutically acceptable carrier comprises one or more agents selected from the group consisting of a solvent, a buffer, an emulsifier, a suspending agent, a disintegrating agent, and a dispersing agent. , Binders, excipients, stabilizers, chelating agents, diluents, gelling agents, wetting agents, absorption delaying agents, liposomes and the like.