TW201422812A - Metschnikowia bicuspidate strain, composition containing the strain and use of the strain - Google Patents

Abstract

The present invention provides Metschnikowia bicuspidate strains RS550 and LL58, which are isolated from Macrobrachium rosenbergii, have probiotic characteristics and can be applied to aquaculture. The present invention also provides a composition containing said strains and a use of said strains.

Description

M. cerevisiae strain, composition containing the same, and use of the same

The present invention relates to a microorganism strain, and in particular to a strain of Metococcus, a composition containing the strain, and the use of the strain.

The development of immunostimulants originally originated from therapeutic studies of human cancers. Some immunostimulants have been found to activate macrophages, T cells, B cells, and natural killer cells, thus leading to the death of cancer cells in the body. In aquaculture, immunostimulants have been used in fish and crustaceans to increase disease resistance by increasing non-specific immune responses, which are effective in controlling diseases. These substances are mostly polysaccharides, such as Beta glucan (β-glucan), laminarin, chitin, mannan oligosaccharide (MOS), and lipopolysaccharide (LPS). Studies have shown that fucoidan increases mammalian disease resistance and has a strong relationship with changes in intestinal flora. Studies have also indicated that some immunostimulants have the effect of stimulating changes in the gut microbiota, for example by stimulating the growth of probiotics in the gut to enhance the disease resistance of the animal.

Probiotics are organisms or substances that promote the balance of intestinal microbes. The new concept of probiotics in the near future includes live bacteria, dead cells, bacterial extracts, and even microbial metabolites, which can be called probiotics. In addition to bacteria, there are also bacteria, yeasts, microalgae, etc., usually lactic acid bacteria and Bacillus spp. are the main probiotics. Previous studies have found that lactic acid bacteria will become healthy. The main component of the intestinal flora of fish and mammals.

In the application of probiotics in aquaculture, the study indicated that Lactobacillus plantarum can improve immunity and resistance to Streptococcus sp. .2009.Dietary administration of the probiotic,Lactobacillus plantarum,enhanced the growth,innate immune responses,and disease resistance of the grouper Epinephelus coioides.Fish & Shellfish Immunology 26:691-698.); and in shrimp, Lactobacillus ( Lactobacillus spp. ) as a probiotic to feed grass shrimp against Vibrio harveyi (Phianphak et al., 1999. Probiotic use of Lactobacillus spp. for black tiger shrimp. Penaeus monodon. Journal of Scientific Research Chulalonkorn University 24, 41-51.); feeding Pediococcus acidilactici enhances the immunity of blue prawn ( Litopenaeus stylirostris ) and resists the pathogen Vibrio nigripulchritudo and improves shrimp survival (Castex et Al.,2010.Effect of probiotic Pediococcus acidilactic i on antioxidant defences and oxidative stress of Litopenaeus stylirostris under Vibrio nigripulchritudo challenge.Fish & Shellfish Immunology XXX: 1-10.).

In addition, Taiwan Public Publication No. 201119588, No. 201136527, and No. 201136529 disclose that it will be separated from white peony and peach. Bacillus natto, yeast and lactic acid probiotics from plum, terrestrial animal feed, pickled food, etc. are used as feed additives for water products.

The strains disclosed above are not isolated from water-producing substances and are not cultured. If these strains are used in aquaculture for a long time, they may not be stable and continue to survive. They must be added frequently in the breeding environment, except for poor economic benefits. In addition, it may cause unpredictable impact on the environmental ecology.

The main object of the present invention is to provide a microbial strain suitable for use in an aquaculture environment and having probiotic characteristics, and a composition containing the same and use of the strain.

In order to achieve the foregoing object, the present invention provides a Metschnikowia bicuspidate RS550 deposited at the Hsinchu Food Industry Development Research Institute of Taiwan under the accession number BCRC 920081.

The invention further provides a Metschnikowia bicuspidate LL58 deposited in the Hsinchu Food Industry Development Research Institute of Taiwan under the registration number BCRC 920082.

In one embodiment of the invention, the aforementioned strain is isolated from Macrobrachium rosenbergii .

The invention further provides a Metschnikowia bicuspidate RS550, which is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan, with the accession number BCRC 920081; and the Met yeast strain LL58, deposited in the Hsinchu Food Industry Development Research Institute of Taiwan, the registration number For BCRC 920082, for aquaculture, as a probiotic and for increasing the immunity of shrimp.

The present invention further provides a composition comprising a feed; and at least one microbial strain, wherein the microbial strain is selected from one or both of the strains according to claim 1 or 3.

The aforementioned strain can be stably and continuously survived in a culture environment, and the strain is mixed with the feed and fed, which can effectively increase the immunity of the water product, and can be used as a probiotic for aquaculture.

Detailed construction, features, assembly or use of the present invention will be described in the detailed description that follows. However, it should be understood by those of ordinary skill in the art that the present invention is not limited by the scope of the invention.

The technical contents and features of the present invention will be described in detail below with reference to the accompanying drawings and the accompanying drawings.

Experiment 1: Changes of microbes in the liver and pancreas of shrimp before and after feeding of fucoidan

1. Brown algae polysaccharide feeding freshwater long-legged prawns

The fucoidan polysaccharide was dissolved in sterilized 0.01 M phosphate buffered saline (PBS) and adjusted to a concentration of 300 μg/mL. Macrobrachium rosenbergii , which was domesticated for three days, was divided into treatment group and control group. The treatment group was fed with 100 μL of fucoidan solution, and the control group was fed with PBS solution.

2. Isolation and culture of microorganisms in the liver and pancreas of shrimp

After the shrimps were sacrificed, the whole hepatopancreas and intestines were taken out, soaked in 20 mL of sterile 0.85% NaCl, and the large tissue in the solution was ground with a mortar, and then centrifuged at 2000 rpm, 4 ° C, 10 min. The supernatant was filtered through a sterile evacuation filter bottle and 5.0 μm filter paper. The filtrate was adjusted to 25 mL with a sterile 0.85% NaCl, and concentrated for culture.

Dilute the bacterial solution to an appropriate concentration, spread the bacterial solution evenly onto the Soybean Protein Agar (Secondary Soy agar; TSA) plate by smear, incubate overnight at 28 ° C or longer, and randomly pick before each group. 50 single colonies were taken and 30 single colonies were picked from each treatment group.

3. Extraction of strain DNA

30 colonies were randomly selected from each group, and cultured into a bacterial solution by Tryptic soy broth (TSB). 1.5 mL of the bacterial solution was taken into a centrifuge tube, and centrifuged at 14,000 rpm for 5 minutes to remove the supernatant. The solution was suspended in 567 μL of TE buffer, and then added with 20 μL of lysozyme in a 37 ° C water bath for 2 hours, after which 3 μL of proteinase solution and 30 μL of 10 were added. The % SDS solution was uniformly mixed and placed in a 37 ° C water bath for 1 hour or more until the solution was clear.

Then add 100 μL of 5M NaCl solution, mix well and then add 80 μL of CTAB-NaCl solution. This solution must be preheated at 65 °C. Mix well and place in a 65 ° C water bath for 15 minutes, add 700 μL of chloroform-IAA solution, mix well, centrifuge at 14000 rpm for 10 minutes, then take the supernatant 600 μL, add RNase 3 μL, and place at 37 ° C water. Act in the bath for 30 minutes. Add 600 μL of dichloromethene, mix well, centrifuge at 14000 rpm for 10 minutes, then take 500 μL of the supernatant, then add 350 μL of 2-propanol, mix well, and let it stand at room temperature for 15 minutes, centrifuge at 14000 rpm for 10 minutes. Remove the supernatant, add 0.5 mL of frozen 75% alcohol, rinse the pellet, centrifuge at 14000 rpm for 10 minutes, remove the supernatant and air dry, add 30 μL of sterile water, slowly shake and dissolve, and put in a 55 ° C water bath. In 10 minutes.

4. Amplification of 16S and 18S rDNA and analysis of agar colloidal electrophoresis

Polymerase chain reaction (PCR) amplification of 16S/18SR (SEQ ID NO: 2, 3) by 16S rDNA primer pair 8F/1510R (SEQ ID NO: 1, 2) and 18S rDNA primer pair 16S and 18S rDNA, conditions are as follows: 10 × PCR buffer 2.5 μL, 10 mM dNTP 0.5 μL, 10 μM primer pair 0.5 μL, sample DNA 2 μL, Tag DNA polymerase 0.25 μL, and make up the volume of sterile water to 25 μL in the PCR tubule. The PCR reaction was carried out by polymerase chain reaction reactor. The reaction conditions were as follows: 94 ° C for 5 minutes, followed by 94 ° C for 1 minute, 51.5 ° C for 1 minute and 30 seconds, 72 ° C for 1 minute for 30 cycles, and finally 72. The reaction was carried out for 10 minutes at °C, and the reaction was stopped at 4 °C.

5 μL of the PCR product was placed in a horizontal electrophoresis tank on 2% agar (agarose), 0.5× TAE buffer solution was added, electrophoresis was carried out at 100 V for 30 minutes, and stained with ethidium bromide (EtBr). The calibration was performed with 100 to 3000 bp as a control, and the electrophoresis colloid was photographed with a colloidal imaging system.

5. Sequence analysis of 16S and 18S rDNA

The results of the PCR products were sequenced at the sequencing center of Yangming University, and the results of the obtained nucleic acid sequences were compared with the database of the National Center for Biotechnology Information (NCBI) to find the closest sequence. Know the species.

6. Composition of microorganisms in the liver and pancreas of shrimp before and after feeding of brown algae polysaccharide

The results of identification and analysis of 16S rDNA and 18S rDNA composed of bacteria in the liver and pancreas of the control group and the treatment group were summarized in Table 1 and Table 2.

As shown in Table 1, the 0th day is a blank group without any treatment of the shrimp, wherein the composition of the bacteria in the hepatopancreas is as follows: G+ is Lactococcus garvieae , Enterococcus sp. , Cellulomonas sp. , Oerskovia sp. and Agromyces italicus , G - for Escherichia coli and Thermus scotoductus . On the third day after feeding PBS, the strains of the liver and pancreas of the shrimp were as follows: G+ was Cellulomonas sp. , A.italicus , O.turbata and Brevibacterium sp. , G- was E.coli , Enterobacter aerogenes , Citrobacter sp. , Flavobacterium sp ., Aeromonas hydrophila and T.scotoductus. After the fifth day, the strains of the liver and pancreas of the shrimp were as follows: G+ was L. garvieae , Agromyces sp. , Microbacterium sp. and O.turbata .; G- was E. coli and A. hydrophila ; Day 10, shrimp liver The strains of the pancreas are as follows: G+ is E.faecalis , O.turbata , A.italicus Microbacterium sp. and L.garvieae ; G-mainly E.coli , the separation rate is 66.7% (20/30), in addition A.hydrophila and C.freundii . The above results showed that the blank group of shrimp had liver-pancreatic pancreas originally associated with pathogenic bacteria, including L. garvieae , A. hydrophila and C. freundii .

As shown in Table 2, on the third day after feeding the brown algae polysaccharide, the strains of the liver and pancreas of the shrimp were as follows: yeast Metschnikowia bicuspidate ; G+ only L. garvieae ; G- was Pantoea sp. , Enterobacter sp ., C. freundii , E .coli , Shewanella sp. and; on day 5, the species of shrimp liver and pancreas are as follows: yeast M.bicuspidate ; no G+ strain; G- are E.coli , Acinetobacter baumannii , Cupriavidus sp. , Uncultured Geobacter sp. And T.scotoductus ; on the 10th day, the strains of the liver and pancreas of shrimp were as follows: G+ were L. garvieae , A.italicus , Cellulomonas sp. and Oerskovia sp. , respectively, G- Pantoea sp ., Enterobacter sp. and Klebsiella On the 15th day, the strains of the liver and pancreas of the shrimp were as follows: yeast M. bicuspidate ; G+ only L. garvieae , G- respectively A. hydrophila , Pseudomonas saccharophila , E. aerogenes and C. freundii .

The above results were compared between the treatment group and the control group. The bacterial phase in the liver and pancreas of the treated group and the control group was as shown in Table 3. After feeding the brown algae polysaccharide, except for the fifth day, on the third day and the tenth day, although the pathogen L. was still present in the liver and pancreas of the shrimp . Garvieae appeared, but the isolation rate decreased; in addition, G-pathogen C. freundii was measured on the third day after feeding, and G-pathogens A. hydrophila and C. freundii were not found at other times. In addition, the yeast M.bicuspidate appeared after feeding the brown algae polysaccharide, and the highest separation rate on the fifth day was 23.3% (7/30).

All the M.bicuspidate isolates isolated from the above were subjected to 19 kinds of carbohydrate metabolism analysis, which can be divided into two groups. The same ratio of carbohydrate metabolism between the two groups was 95%; and then 18S rDNA sequence alignment, hope to select metabolism and Strains that have a large difference in physiological functions may be evaluated for subsequent probiotics. The results showed that the LL58 and RS550 strains in all isolates had the same ratio of carbohydrate metabolism of 95%, but they were different from the results of the previously published Metschnikowia spp. (Table 4); In addition, the similarity between the 18S rDNA sequences in all isolates (99.5~100%), the similarity of the two strains was 99%, the difference was large.

Based on the above results, two strains of LL58 and RS550 (Fig. 1) were selected for the evaluation of the following probiotics.

Experiment 2: Evaluation of isolates as probiotics

1. Toxicity test of M.bicuspidate LL58 and RS550

Two isolates M.bicuspidate LL58 and RS550 appearing in the hepatopancreas of shrimp after treatment with fucoidan and Lactobacillus plantarum ATCC10012 from mammals and confirmed to be probiotic strains were used as controls, and high doses of bacteria were injected by injection. Toxicity test of three strains on shrimp.

Respectively L.plantarum ATCC10012, M.bicuspidate LL58 and RS550 was inoculated into brain heart infusion culture medium (Brain heart infusion broth; BHIB, Difco) , and TSB (Difco), placed in an incubator (FIRSTEK SCIENTIFIC, S300R) at 28 ℃ , 130 rpm shaking culture to late log phase. Take 15 mL of the bacterial solution, centrifuge at 5000 rpm, 4 ° C for 10 minutes, resuspend the bacterial solution with PBS (pH = 7.56, 420 ± 5 m Osm / kg), and adjust the concentration of the bacterial solution to 1 × 10 ⁹ CFU / mL. The bacterial solution was extracted with a 1 mL syringe (25G), and 100 μL of the bacterial solution was injected into the first section of the abdominal cavity of the shrimp, and the number of shrimp deaths was counted every 12, 24, 48, and 72 hours. The mortality rate is calculated as follows: mortality (%) = (only deaths in the experimental group - non-specific deaths) / (total - non-specific deaths) × 100%. The dead within 12 hours was identified as a non-specific death.

The results are shown in the following Table V, as a control L.plantarum shrimp lethality rate of 87%, respectively, and RS550 lethal M.bicuspidate LL58 shrimp from 12% and 22%.

N, the number of shrimp used in each set of experiments.

2. M. bicuspidate LL58 and RS550 extracellular fluid co-cultured with pathogenic bacteria

Respectively, three bacterial extracellular fluid L.plantarum, M.bicuspidate LL58 RS550 and co-cultured with bacteria to observe three bacteriostatic effect. The L.plantarum, M.bicuspidate LL58 RS550 and inoculated into MRS medium (Oxoid), BHIB (Difco), and TSB (Difco), placed in an incubator at 28 ℃, 130 rpm shaking culture to different growth periods. Take 40 mL of the bacterial solution, centrifuge at 5000 rpm, 4 ° C for 10 minutes, take 40 mL of the supernatant, and dialyze with phosphate buffer (Phosphate buffer; PB, pH = 7.0) at 4 ° C for 18 to 24 hours, then The supernatant was concentrated in a freeze dryer (Labconco) and reconstituted with 0.85% NaCl to carry out an bacteriostatic test.

The pathogenic bacteria L. garvieae S99 isolated from the liver and pancreas of shrimp and two pathogenic bacteria Aeromonas hydrophila and A. veronii ATCC 9071 from the center of the strain were inoculated into BHIB (Difco) and TSB (Difco), and placed in an incubator. The medium was shaken at 28 ° C and 130 rpm. Take an appropriate amount of bacterial solution, centrifuge at 5000 rpm, 4 ° C for 10 minutes, wash twice with 0.85% NaCl, then dissolve back with low nutrient medium (Poor-nutrient broth; PB), and adjust the bacterial solution to an OD ₆₀₀ of 0.01. The experiment was divided into three groups. In the 96-well plate, the blank group was added with 90 μL of PB medium and 10 μL of blank medium. The control group was added with 90 μL of bacterial solution and 10 μL of blank medium. The experimental group was added with 90 μL of bacterial solution and 10 μL of each. The supernatant of the bacteria to be tested was cultured at 28 ° C for 12 hours, and the absorbance was measured every hour by an enzyme-linked immunosorbent assay (ELISA) reading instrument. The obtained absorbance value was selected based on the control group, and the bacteria were observed at different time points of the growth period to observe the bacteriostasis of each test bacteria.

Results As shown in the second to fourth figures, the three strains were bacteriostatic in the log phase, late log phase, and stationary phase extracellular fluid, L. The extracellular fluids of plantarum and M.bicuspidate LL58 from shrimp also inhibited the growth of pathogenic bacteria A. hydrophila and A. veronii . The former had the best effect on the extracellular fluid in the late log phase, and the inhibitory concentration was 0.88 mg/mL, the latter is the best in the stable phase, the inhibitory concentration is 3.03 mg / mL; the other strain of shrimp isolate M.bicuspidate RS550 on the growth of three pathogenic bacteria L. garvieae and two strains of Aeromonas All of them have an inhibitory effect, and the effect of the extracellular fluid in the late log phase is the best, and the inhibitory concentration is 0.076 mg/mL.

3. Extracellular enzymes of pathogenic bacteria co-cultured with M.bicuspidate LL58 and RS550

Three strains of pathogenic bacteria in different growth stages were co-cultured with two probiotics to observe the antibacterial effect. After separating the L. garvieae S99, A. hydrophila and A. veronii ATCC 9071 from the extracellular solution according to the above method, M.bicuspidate LL58 and RS550 were placed in a 96-well plate and cultured at 28 ° C for 16 hours. The absorbance was read by an ELISA reader, and the bacteriostasis of each test bacteria was observed.

Results As shown in the fifth and sixth figures, M.bicuspidate LL58 was not inhibited by three pathogenic bacteria, and M.bicuspidate RS550 was inhibited by extracellular fluid of L. garvieae at any stage.

4. Changes in phenoloxidase activity of shrimp after feeding M.bicuspidate LL58 and RS550

After 5 days of continuous feeding of freshwater long-legged prawns with M.bicuspidate LL58 and RS550 (10 ⁶ CFU/mL), the hemolymph of the shrimp was taken and centrifuged at 3200 rpm for 5 minutes at 4 ° C (general-purpose high speed). After refrigerating the centrifuge, HERMLE, Z326K), the supernatant, ie the plasma sample, was collected; the blood cell pellet was added to 200 μL of PBS (0.01 M) buffer to suspend the cells, and then centrifuged at 12,000 rpm for 4 minutes at 4 ° C for 30 minutes. After rupture, the supernatant is taken as hemocyte lysate supernatant (HLS).

The activity of phenoloxidase (PO) was measured by L-DOPA (L-3,4-dihydroxyphenyl-alanine; Sigma, D9628). 50 μL of HLS was placed in a 96-well microplate, and 50 μL of 1 mg/mL tryptic (trypsin, Sigma) or 10 mM PBS was added to each well. After catalysis at 37 ° C for 15 minutes, immediately add 200 μL of fresh. The prepared 1.6 mg/mL L-DOPA/10 mM PBS was used as the substrate, and the absorbance was measured by an ELISA reader at a wavelength of 490 mm. The OD ₄₉₀ per minute was taken within the first five minutes. The maximum value represented by the PO represented PO. The maximum activity change per unit time, the OD ₄₉₀ change per unit time is 0.001 to 1 U, and the PO activity is expressed as ΔU/Δt/mg.

In the experiment, the PO activity measured by the sample without the addition of tryptone treatment is the naturally activated PO content of the sample (in plasma or blood cells), expressed as PO _S ; the PO _S value in the plasma can represent the release particle of the blood cell in the plasma. PO activity, the larger the value, the stronger the activity. The PO activity measured by adding tryptone-treated sample (in plasma or blood cell) can be regarded as the total amount of proPO originally contained in the sample, expressed as PO _T ; the PO _T value in plasma can also represent the release particle of blood cells. Role, the PO _T value in the blood cell represents the amount of proPO production.

The results of the assay are shown in Figure 7. The plasma concentrations of PO _S (0.0633 U/min/mg) and PO _T (1.5733 U/min/mg) in the M.bicuspidate RS550 feeding group were compared with the control group fed with PBS (0.0667 U/). There was no difference between min/mg and 3.4533 U/min/mg. As for the PO _S and PO _T (1.0353 U/min/mg and 1.5900 U/min/mg) in the blood cells, it was higher than the control group (0.0700 U/min/mg and 0.5567 U/min/mg); in the blood cells of M.bicuspidate LL58 feeding group, PO _S and PO _T and plasma PO _T (0.0667 U/min/mg, 0.5667 U/min/mg and 1.6000 U/min/mg) There was no difference in the control group (0.0700 U/min/mg, 0.5567 U/min/mg, and 3.4533 U/min/mg), while plasma PO _S (0.1800 U/min/mg) was higher than the control group (0.0667 U/min/). mg); M.bicuspidate LL58 after mixing and RS550 shrimp fed _{group, PO S (0.0933 U / min} / mg) and plasma _{PO T (2.5600 U / min /} mg) were fed with PBS control group (0.0667 U There was no difference between /min/mg and 3.4533 U/min/mg. As for the PO _S and PO _T (3.5233 U/min/mg and 4.9467 U/min/mg) in the blood cells, it was higher than the control group (0.0700 U/min/mg). And 0.5567 U/min/mg).

Comparing the PO activity activation of the experimental group and the control group, as shown in Table 6, it can be found that the plasma and intracellular activation ratio (0.0402 and 0.6511) of the M.bicuspidate RS550 feeding group were higher than the control group (0.0193 and 0.1257); There was no difference in the intracellular activation ratio (0.1177) and the control group (0.1257) in the M.bicuspidate LL58 feeding group, but the plasma activation ratio (0.1125) was higher than that in the control group (0.0193); the plasma and blood cells of the two probiotic feeding groups were mixed. The internal activation ratio (0.0364 and 0.7123) was higher than that of the control group (0.0193 and 0.1257).

Because the proPO activation system begins to affect and enhance other defense activities while releasing the hemorrhagic ball into the plasma. From the PO activity ratio results, it was found that the plasma PO _S and PO _T of the shrimp fed with M.bicuspidate RS550 for 5 days did not differ from the control group, but the PO _{T in the} blood cells was significantly higher than that of the control group, and the result showed that M.bicuspidate was fed . RS550 can improve the immunity of shrimps. It is speculated that RS550-fed shrimp blood cells can release more and more active proPO activation system when infection occurs. As for M.bicuspidate LL58 feeding group, plasma PO _{S is} higher than control group. , indicating that the immunity of the shrimp has been activated to a certain extent; while the PO _T in the blood of the mixed feeding group is significantly higher than that of the control group, and higher than the M.bicuspidate RS550 feeding group, showing mixed M.bicuspidate RS550 and LL58 feeding. Shrimp can better enhance immunity.

In summary, feeding two probiotics, M.bicuspidate RS550 and LL58, can increase the activity of proPO activation system in plasma and blood cells. Since the performance in blood cells is more obvious than plasma, it is speculated that feeding two probiotics can moderately enhance shrimp immunity, but should Will not cause excessive inflammation of the shrimp.

5. Susceptibility testing of shrimp after feeding M.bicuspidate LL58 and RS550

Two probiotics, M.bicuspidate LL58 and RS550, were inoculated into BHIB (Difco) and TSB (Difco), respectively, and placed in an incubator at 28 ° C, 130 rpm to culture until the late log phase. Take an appropriate amount of bacterial solution, centrifuge at 5000 rpm, 4 ° C for 10 minutes, resuspend the bacterial solution with PBS (pH = 7.56, 420 ± 5 m Osm / kg), and adjust the bacterial solution to a concentration of 1 × 10 ⁷ CFU / mL . Use a 1 mL empty syringe to attach a thin hose, and use a hose to contact the shrimp mouth. First, let the shrimp mouthpiece suck the tube, inject 100 μL of the bacterial solution, and feed for five days.

The pathogenic bacteria L. garvieae was inoculated into BHIB (Difco), placed in a 28 ° C incubator, and incubated at 130 rpm until late in the log phase. Take an appropriate amount of bacterial solution, centrifuge at 5000 rpm, 4 ° C for 10 minutes, resuspend the bacterial solution with PBS (pH = 7.56, 420 ± 5 m Osm / kg), and adjust the bacterial solution to a concentration of 5 × 10 ⁹ CFU / mL 100 μL of bacterial solution was injected into the first section of the abdominal cavity of the shrimp, and the number of shrimp deaths was counted every 12, 24, 48, and 72 hours. According to the aforementioned mortality calculation formula, the specific mortality rate is converted.

The experiment was divided into eight groups, and each group of experiments used 40 shrimps. The control group were fed with PBS, one group was injected with PBS, the other group was injected with pathogenic bacteria L. garvieae , and the experimental group was fed with probiotic M. bicuspidate LL58, one group was injected with PBS, and the other group was injected with pathogen L. garvieae . the other two groups were fed M.bicuspidate RS550, a group injected with PBS, to another group of pathogens L.garvieae injection of the last two and fed M.bicuspidate LL58 RS550 mixing 50 μL of each of the bacteria, one group injected with PBS, the other One group was injected with the pathogenic bacteria L. garvieae .

The results are shown in Table 7. The PBS feeding group died after infection (100% mortality), while the M.bicuspidate RS550 feeding group and the M.bicuspidate LL58 feeding group had 29% and 27% mortality, respectively. Mixed M.bicuspidate The mortality rate of shrimp in the RS550 and LL58 feeding groups was 0%.

From the above experimental results susceptibility, shrimp survival M.bicuspidate RS550 and M.bicuspidate LL58 fed group were fed the same group, the display of the two isolates resistance enhancing effect equivalent shrimp, then mixing two isolates shrimp group after feeding There is no death, indicating the best improvement.

The results of the above-mentioned experiments are shown in Table 8. The M.bicuspidate RS550 and LL58 isolates obtained after the fucoidan fed the shrimp were analyzed by probiotics. The two isolates inhibited the pathogenic bacteria and did not (or weakened) the shrimp. ) Toxicity and susceptibility to reducing susceptibility (promoting resistance) can be used as a probiotic for shrimps in aquaculture.

ND: not detected

Experiment 3: Feeding shrimp with M.bicuspidate LL58 and RS550

1. Production of feed

The blank feed (provided by Professor Zheng Wenteng of Pingtung University of Science and Technology) and PBS, alginic acid (Alginic W201502, Sigma), M.bicuspidate LL58 bacterial solution, RS550 bacterial solution or two strains of the mixture, in a weight ratio of 1:1 After mixing (W/V), remove the needle with a 3 mL empty syringe to extrude the feed, air dry for 30 minutes, and then cut the feed to a size of about 3 mm, as shown in Figure 8. Dissolve 10 feeds in 10 mL of 0.85% NaCl for 10 times serial dilution, then take 100 μL of 100 μL dilution plates at 10 ³ , 10 ⁴ , and 10 ⁵ , and incubate at 28 ° C for 17 hours. Multiply the number of colonies by the dilution factor and push back the average number of bacteria per feed.

The blank feed powder was added to the feed prepared from alginic acid, and the dissolution time was tested after the water was poured into the pool. The results showed that the feed granules added with the alginic acid solution began to spread after about 15 minutes in the water, and the unadded control group was spread at 5 minutes. Subsequent to the feed used in the experiment, alginic acid was added to ensure that the shrimp could eat the whole feed. Feed the feed of probiotics approximately 3 mm in size When eating shrimp, the feed can be successfully found and eaten by the shrimp within 15 minutes.

In order to accurately grasp the number of viable bacteria per oral administration of shrimp, the average number of bacteria per feed was 10 ⁴ CFU/mL. After the feed was stored at 4 ° C, the number of bacteria in each feed was maintained at 10 ⁴ CFU/mL for 5 consecutive days (Table 9).

2. Susceptibility testing of shrimp after feeding

The pathogenic bacteria L. garvieae was inoculated into TSB (Difco), placed in a 28 ° C incubator, and cultured at 130 rpm until the log phase. Take an appropriate amount of bacterial solution, centrifuge at 6000 rpm, 4 ° C for 10 minutes, and then resuspend the bacterial solution in PBS (pH = 7.56, 420 ± 5 m Osm / kg), and adjust the concentration of the bacterial solution to 1 × 10 ⁹ CFU / mL spare.

Experimental group feeds containing alginic acid, LL58, RS550, and mixed LL58 and RS550 were prepared, and the control group feed was not wrapped with any substance. Continuous feeding of shrimp on the 5th, on the 6th day, oral infection with the pathogen L. gariveae (10 ⁹ CFU / mL), using a 1 mL empty syringe to connect a thin hose, suck the bacterial liquid, and then touch the hose to the shrimp Oral device, when the shrimp bites the hose, then inject 100 μL of the bacterial liquid, observe the number of shrimp deaths every 12, 24, 48, and 72 hours, and obtain the specific mortality according to the above formula. This susceptibility experiment was performed twice in total, and the results are shown in Tables 10 and 11, respectively.

N=shrimp only

The mortality rate of the first susceptibility test was 50% in the control group, 25% in the alginic acid group, 11.5% in the LL58 group, 12.5% in the RS550 group, and 11.1% in the mixed group. The mortality rate of the second susceptibility test was 46.7% in the control group, 23.1% in the alginic acid group, 11.1% in the LL58 group, 11.8% in the RS550 group, and 11.8% in the mixed group.

N=shrimp only

From the results of two feeding experiments, it can be seen that whether feeding M. bicuspidate LL58, RS550 or mixed two isolates alone can effectively reduce susceptibility and mortality.

3. Phenol oxidase activity test of shrimp after feeding

The phenol oxidase activity test method is as described in Experiment 2. Five days after the continuous feeding of the probiotic-containing feed, the shrimp blood lymphocytes were taken before the infection of the pathogenic bacteria and on the second day after the infection, and the PO _{T in the} blood cells and plasma were measured, and the shrimps in each group were 10 tails. Results As shown in the ninth figure, the PO _T activities of the alginic acid group, the LL58 group, the RS550 group and the mixed group of shrimps after continuous feeding of probiotics for 5 days were 83.76, 113.85, 115.22 and 64.49 U/min/mg, respectively. The PO _{T of the} group of shrimps was higher than that of the control group of 29.6 U/min/mg. The PO _T results in the blood of each group of shrimps were found to be higher than the control group (77.05 U/min/mg) except the LL58 group (77.05 U/min/mg), which were higher than the control group, respectively. The internal PO _T was 21.19 U/min/mg, the alginic acid group was 117.1 U/min/mg, and the mixed group was 87.4 U/min/mg.

The susceptibility test of the shrimp was carried out after the shrimp containing the probiotics LL58 and RS550, the two strains mixed, the alginic acid and the control group were continuously fed for 5 days, and the results of measuring the PO _T after 2 days of infection were as shown in the tenth figure. The PO _{T in the} control group was 67 U/min/mg, 91.89 U/min/mg in the blood cells, 74.8 U/min/mg in the LL58 plasma, 92.25 U/min/mg in the blood cells, and 121.96 U/min in the RS550 plasma. /mg, 141.09 U/min/mg in the blood cell; plasma 62.35 U/min/mg in the alginic acid group, 77.48 U/min/mg in the blood cell, 89.57 U/min/mg in the plasma of the two probiotic mixed groups, blood cells The inside is 37.96 U/min/mg.

When the shrimp being L.garvieae infection, releasing blood cell degranulation fed group than the control group cells, and can continue to generate the proPO blood cells, and a control group maintained higher than or equivalent to the amount synthesized. From the results, it can be speculated that the feeding isolate can indeed enhance the immunity of the shrimp.

4. Glutathione peroxidase activity test of shrimp after feeding

Glutathione peroxidase (GPx) activity assay was performed using a commercially available test kit (Glutathione peroxidase assay kit, Fortress Diagnostics) to determine GPx activity in shrimp plasma. The extraction method of plasma extract was as described in previous experiments. .

The results of GPx activity test in shrimp blood cells after 5 days of continuous feeding were as shown in the eleventh figure. The alginic acid group, LL58 group and RS550 group were not significantly different from the control group, but the GPx activity of the mixed group was 6776.71 U/cells. Higher than the control group. The GPx activity measured two days after infection, except for the mixed group activity 4837.23 U/cells was still higher than the control group, the other four groups of GPx activity remained the same as the control group. The activity of each group measured on the 4th day after infection was reduced compared with the GPx activity measured two days after infection. From the results, it can be speculated that feeding probiotics can also enhance the antioxidant capacity of shrimps.

It is to be understood that the foregoing detailed description is merely illustrative of the nature and features of the invention, and those of ordinary skill in the field of the invention </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

The first image is a scanning electron micrograph of M.bicuspidate LL58 and M.bicuspidate RS550 in the late logarithmic growth phase.

The second picture shows the experimental results of the inhibitory effect of L. plantarum extracellular fluid on the growth of three pathogenic bacteria.

The third picture shows the inhibitory effect of M.bicuspidate LL58 extracellular fluid on the growth of three pathogenic bacteria.

The fourth picture shows the experimental results of the inhibitory effect of M.bicuspidate RS550 extracellular fluid on the growth of three pathogenic bacteria.

The fifth panel is the experimental results of the inhibition of the growth of M.bicuspidate LL58 by the extracellular fluid of three pathogenic bacteria.

The sixth figure is the experimental results of the inhibition of the growth of M.bicuspidate RS550 by the extracellular fluid of three pathogenic bacteria.

The seventh picture shows the plasma and phenol oxidase activity PO _S and PO _T test results of freshwater long-legged prawns after feeding probiotics.

The eighth picture shows the preparation of feed pellets after living bacteria

The ninth figure shows the results of PO _T measurement of plasma and total phenol oxidase activity in freshwater long-legged prawns after five days of continuous feeding of different feeds.

The tenth figure shows the results of PO _T measurement of plasma and blood phenol oxidase activity in freshwater long-legged prawns fed with different feeds two days after Lacotoccus garvieae susceptibility test.

The eleventh figure shows the results of determination of glutathione peroxidase activity in the blood cells of freshwater long-legged prawns fed different feeds 2 days and 4 days after the susceptibility test with Lacotoccus garvieae .

<110> Soochow University

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Claims (10)

A Metschnikowia bicuspidate RS550 is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan under the registration number BCRC 920081. The strain of claim 1, which is isolated from Macrobrachium rosenbergii . A strain of Metschnikowia bicuspidate LL58 is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan under the registration number BCRC 920082. The strain of claim 1, which is isolated from Macrobrachium rosenbergii . A strain according to claim 1 or 3, for use in aquaculture. A strain according to claim 1 or 3, which is used as a probiotic. A strain according to claim 1 or 3, which is for use in increasing immunity of shrimp. A composition comprising: a feed; and at least one microbial strain, wherein the microbial strain is selected from one or both of the strains according to claim 1 or 3. The composition of claim 1 further comprising a brown alginic acid. The composition of claim 1 or 2, wherein the number of bacteria in the composition is from 10 ⁴ to 10 ⁵ CFU/mL.