CN114223777B - Composite microecological preparation for inhibiting pathogenic vibrio for breeding stichopus japonicus - Google Patents

Abstract

The invention discloses a compound microecological preparation for inhibiting pathogenic vibrio for culturing stichopus japonicus, which is prepared by mixing rhodopseudomonas palustris, clostridium butyricum, oak leaf extract and aloe leaf extract as feed additives, wherein the addition amount of each gram of feed is 10% of rhodopseudomonas palustris ⁵ ~10 ⁷ cfu, clostridium butyricum 10 ⁵ ~10 ⁷ cfu, 1-2 mg of oak leaf extract and 4-8 mg of aloe leaf extract; the oak leaf extract and aloe leaf extract are prepared according to the following method: respectively cleaning oak leaf and aloe leaf with distilled water, and drying in shade at 25deg.C; homogenizing and crushing oak leaves and aloe leaves by a homogenizer respectively, and adding an ethanol solution with the volume fraction of 65-85%, wherein the feed-liquid ratio is 0.05-0.1 g/mL; leaching for 1-2.5 h at normal temperature under the action of ultrasonic waves with the working frequency of 40 KHZ and the power of 80W; loading the leaching solution into an ultrafiltration membrane device, filtering to remove impurities under the pressure of 0.06Mpa, concentrating under reduced pressure, and lyophilizing to obtain lyophilized powder.

Description

Composite microecological preparation for inhibiting pathogenic vibrio for breeding stichopus japonicus

Technical Field

The invention belongs to the fields of microbial engineering technology and echinoderm disease control, and particularly relates to a composite microecological preparation for inhibiting pathogenic vibrio for stichopus japonicus cultivation.

Background

Radix stichopae japonicaeApostichopus japonicus) Belonging to Echinodermata and Stichopus japonicus class, which is rich in various natural active substances such as protein, mineral substances, vitamins, etc., has extremely high nourishing and medicinal value, and is rapidly developed in the current stichopus japonicus culture industry. The beancurd sheet syndrome is a common disease in the stichopus japonicus culture process, and has the characteristics of strong outbreak, high infection speed, high death rate and the like. It has been found that pathogenic vibrio such as vibrio splendidus, vibrio harveyi, vibrio parahaemolyticus and the like are main pathogenic bacteria causing stichopus japonicus 'skin syndrome'. Due to the problems of food safety, environmental protection, drug resistance and the like, antibiotic drugs for inhibiting pathogenic vibrio are gradually abandoned by aquaculture industry. Therefore, development of green pollution-free antibiotic substitutes for inhibiting pathogenic vibrio is needed to improve immune function and disease resistance of stichopus japonicus.

Rhodopseudomonas palustris contains abundant proteins, vitamins, trace elements and physiologically active substances, can degrade nitrite, sulfide and other toxic substances in water, simultaneously releases enzymes with disease resistance, can promote proliferation of beneficial microorganisms, and can inhibit proliferation of a plurality of pathogenic bacteria. Clostridium butyricum is an anaerobic bacillus with an endophytic spore structure, is beneficial bacteria for regulating intestinal microecological balance, and the main metabolite butyric acid is a main nutrient substance for regeneration and repair of intestinal cells, so that invasion and colonization of pathogenic bacteria can be prevented in intestinal tracts, intestinal flora disturbance can be corrected, and the microecological balance in a host body can be regulated. Meanwhile, clostridium butyricum can produce substances such as vitamins, protease, amylase, cellulase and the like in the intestinal tract, and has good nutrition and health care effects on organisms. The oak leaves and the aloe leaves have the characteristics of naturalness, nutrition, small toxic and side effects and the like. Meanwhile, the oak leaves and the aloe leaves also have good in-vitro antibacterial capability, not only have an inhibition effect on pathogenic microorganisms, but also can effectively improve the anti-stress capability and disease resistance and restoration capability of organisms.

However, there has been no report so far on a complex microecological preparation inhibiting pathogenic vibrio for cultured stichopus japonicus in which rhodopseudomonas palustris, clostridium butyricum, tussah leaves and aloe She Geli are combined to increase biological potency.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a composite microecological preparation for inhibiting pathogenic vibrio for stichopus japonicus cultivation.

The technical scheme of the invention is as follows: a composite microecological preparation for inhibiting pathogenic vibrio for culturing stichopus japonicus is characterized by comprising rhodopseudomonas palustrisRhodopseudomonas palustris) Clostridium butyricumClostridium butyricum) Mixing oak leaf extract and aloe leaf extract to obtain feed additive, wherein the additive amount of each gram of feed is rhodopseudomonas palustris 10 ⁵ ~10 ⁷ cfu, clostridium butyricum 10 ⁵ ~10 ⁷ cfu, 1-2 mg of oak leaf extract and 4-8 mg of aloe leaf extract; the oak leaf extract and the aloe leaf extract are prepared according to the following method: respectively cleaning oak leaf and aloe leaf with distilled water, and drying in shade at 25deg.C; homogenizing and crushing oak leaves and aloe leaves by a homogenizer respectively, and adding an ethanol solution with the volume fraction of 65-85%, wherein the feed-liquid ratio is 0.05-0.1 g/mL; leaching for 1-2.5 h at normal temperature under the action of ultrasonic waves with the working frequency of 40 KHZ and the power of 80W; loading the leaching solution into ultrafiltration membrane device, filtering under 0.06Mpa to remove impurities, concentrating under reduced pressure, and lyophilizing to obtain lyophilized powder, namely oak leaf extract and aloe leaf extract.

The optimal technical proposal is that rhodopseudomonas palustris 10 is added into each gram of feed ⁶ cfu, dineClostridium sour 10 ⁶ cfu, oak leaf extract 1.5mg, aloe leaf extract 6 mg.

The rhodopseudomonas palustris is prepared according to the following steps:

step 1: the viable count of 0.1 g is 0.5X10 ¹⁰ ~10 ¹⁰ Fully dissolving cfu/g rhodopseudomonas palustris freeze-dried powder microbial inoculum in 1 ml sterile distilled water, and streaking and activating strains on a solid culture medium, wherein the solid culture medium comprises 15.0 g/L tryptone, 5.0 g/L soytone, 5.0 g/L sodium chloride and 20 g/L agar, and pH 7.5-8.0; culturing in an anaerobic incubator at the illuminance of 2000-3000 lux and the temperature of 30-35 ℃ until red single colonies appear on a plate;

step 2: selecting red single bacterial colony, inoculating the red single bacterial colony into a rhodopseudomonas palustris liquid culture medium, wherein the culture temperature is 30-35 ℃, the illumination intensity is 2000-3000 lux, and carrying out stationary culture in an anaerobic incubator for 3-5 days, wherein the liquid culture medium comprises 15.0 g/L of tryptone, 5.0 g/L of soyase and 5.0 g/L of sodium chloride, and pH 7.5-8.0;

step 3: the cells were collected by centrifugation at 6000 rpm for 10 min and resuspended in sterile seawater.

The solid culture medium and the liquid culture medium are also added with the following components: 0.1-0.3 g/L of magnesium chloride, 2-5 mg/L of ferrous sulfate, 0.5-1.5 mg/L of zinc sulfate, 0.1-0.3 mg/L of manganese chloride, 1-4 mg/L of nickel chloride and 0.3-0.8 mg/L of indoleacetic acid.

The clostridium butyricum is prepared according to the following steps:

step 1: 0.1. 0.1 g viable count 0.5X10 ¹⁰ ~10 ¹⁰ Fully dissolving cfu/g clostridium butyricum freeze-dried powder microbial inoculum in 1 ml sterile distilled water, streaking and activating strains on a reinforced clostridium agar culture medium, and culturing in an anaerobic incubator at 37 ℃ until a circular single colony appears on a flat plate; the reinforced clostridium agar medium comprises 10 g/L peptone, 10 g/L beef powder, 3 g/L yeast powder, 5 g/L glucose, 1 g/L soluble starch, 5 g/L sodium chloride, 3 g/L sodium acetate, 0.5 g/L cysteine hydrochloride, 0.15 g/L manganese sulfate, 1.5 g/L dipotassium hydrogen phosphate, 0.25 g/L calcium chloride, 0.65 g/L magnesium sulfate and 15 g/L agar, and has pH value of 6.8-7.2;

step 2: selecting single bacterial colony, inoculating the single bacterial colony into a reinforced clostridium liquid culture medium, and standing the single bacterial colony in an anaerobic incubator for anaerobic culture at 37 ℃ for 1-2 days; the reinforced clostridium liquid culture medium comprises 10 g/L peptone, 10 g/L beef powder, 3 g/L yeast powder, 5 g/L glucose, 1 g/L soluble starch, 5 g/L sodium chloride, 3 g/L sodium acetate, 0.5 g/L cysteine hydrochloride, 0.15 g/L manganese sulfate, 1.5 g/L dipotassium hydrogen phosphate, 0.25 g/L calcium chloride and 0.65 g/L magnesium sulfate, and has a pH value of 6.8-7.2;

step 3: the cells were collected by centrifugation at 6000 rpm for 10 min and resuspended in sterile seawater.

The invention prepares the rhodopseudomonas palustris, clostridium butyricum, oak leaf extract and aloe leaf extract into a compound microecological preparation for the first time by scientific compatibility and is applied to stichopus japonicus cultivation. The rhodopseudomonas palustris and clostridium butyricum belong to facultative anaerobic bacteria species, so that the mutual antagonism between strains can be effectively avoided, on the contrary, butyric acid generated by clostridium butyricum in the metabolic process can neutralize alkaline substances, and the butyric acid can be used as a carbon source for the growth of the rhodopseudomonas palustris to promote the growth of the rhodopseudomonas palustris, and meanwhile, the rhodopseudomonas palustris and the clostridium butyricum can promote the absorption and utilization of the extract of the oak leaves and the extract of the aloe leaves by stichopus japonicus; the active ingredients of the oak leaf extract and the aloe leaf extract can inhibit the mixed bacteria in clostridium butyricum and rhodopseudomonas palustris and provide a carbon source for the growth of the clostridium butyricum and rhodopseudomonas palustris, so that the survival rate of clostridium butyricum and rhodopseudomonas palustris can be improved, and the butyric acid conversion rate of clostridium butyricum can be promoted. The reasonable compatibility of the components of the invention can form a linkage effect, not only can increase the oxygen content of the stichopus japonicus culture water body, but also can promote the immune stimulation effect of a single component and has more obvious targeting property, not only can obviously enhance the immune response of the stichopus japonicus body, but also can act on specific immune cells, thereby selectively inducing the body to form effective immune response aiming at specific antigens, enhancing the nonspecific immune function and disease resistance of the stichopus japonicus, effectively inhibiting the breeding of pathogenic vibrio and having higher biological titer. The invention has important significance for improving the stichopus japonicus culture effect and increasing the economic benefit on the premise of ensuring no pollution, no residue, no drug resistance and no toxic or side effect.

Drawings

FIG. 1 shows colony morphology of rhodopseudomonas palustris (A) and clostridium butyricum (B) in the example of the present invention.

FIG. 2 is a schematic diagram showing the effect of the embodiment of the invention on the cumulative mortality rate of the Vibrio splendidus stichopus japonicus.

FIG. 3 is a schematic diagram showing the effect of the embodiment of the invention on the cumulative mortality of the stichopus japonicus from Vibrio harveyi.

FIG. 4 is a schematic diagram showing the effect of the embodiment of the invention on the cumulative mortality of stichopus japonicus against Vibrio parahaemolyticus.

FIG. 5 is a schematic diagram showing the effect of the embodiment of the invention on the disease condition of the offending stichopus japonicus.

FIG. 6 is a graph showing the effect of the embodiment of the present invention on inhibiting Vibrio splendidus.

FIG. 7 is a graph showing the effect of the embodiment of the present invention on inhibiting Vibrio harveyi.

FIG. 8 is a graph showing the effect of the example of the present invention on inhibiting Vibrio parahaemolyticus.

Detailed Description

Example 1:

the invention relates to a compound microecological preparation for inhibiting pathogenic vibrio for stichopus japonicus cultivation, which is prepared from rhodopseudomonas palustrisRhodopseudomonas palustris) Clostridium butyricumClostridium butyricum) Mixing oak leaf extract and aloe leaf extract as feed additive, and adding rhodopseudomonas palustris 10 per gram of feed ⁵ cfu, clostridium butyricum 10 ⁷ cfu, oak leaf extract 1 mg, aloe leaf extract 4 mg;

the oak leaf extract and the aloe leaf extract are prepared according to the following method: cleaning the outsourced oak leaves and aloe leaves with distilled water respectively, and drying in the shade at 25 ℃; homogenizing and crushing oak leaves and aloe leaves by a homogenizer respectively, and adding 65% ethanol solution with a feed-liquid ratio of 0.05 g/mL; leaching 1 h at normal temperature under the action of ultrasonic wave with working frequency of 40 KHZ and power of 80W; loading the leaching solution into ultrafiltration membrane device, filtering under 0.06Mpa to remove impurities, concentrating under reduced pressure, and lyophilizing to obtain lyophilized powder, namely oak leaf extract and aloe leaf extract.

The rhodopseudomonas palustris is purchased from China industry microbiological culture collection center with the preservation number of CICC23812 and is obtained by expanding culture according to the following steps:

step 1: freeze-dried powder of 0.1 g rhodopseudomonas palustris (viable count 0.5X10) ¹⁰ ~10 ¹⁰ cfu/g) is fully dissolved in 1 ml sterile distilled water, strains are streaked and activated on a solid culture medium, and are cultured in an anaerobic incubator for 3-5 days under the conditions of illuminance of 2000 lux and temperature of 30 ℃, so that red single colonies appear on a plate; the solid culture medium comprises 15.0 g/L of tryptone, 5.0 g/L of soybean peptone, 5.0 g/L of sodium chloride, 20. 20 g/L of agar, 0.1-0.3 g/L of magnesium chloride, 2-5 mg/L of ferrous sulfate, 0.5-1.5 mg/L of zinc sulfate, 0.1-0.3 mg/L of manganese chloride, 1-4 mg/L of nickel chloride and 0.3-0.8 mg/L of indoleacetic acid, and pH 7.5-8.0;

step 2: selecting red single colonies, inoculating the red single colonies into a rhodopseudomonas palustris liquid culture medium, culturing at the temperature of 30 ℃ and the illumination intensity of 2000 lux, and performing stationary culture in an anaerobic incubator for 3-5 days, namely culturing until strains proliferate to logarithmic phase and bacterial liquid turns to dark red; the liquid culture medium comprises 15.0 g/L of tryptone, 5.0 g/L of soybean peptone, 5.0 g/L of sodium chloride, 0.1-0.3 g/L of magnesium chloride, 2-5 mg/L of ferrous sulfate, 0.5-1.5 mg/L of zinc sulfate, 0.1-0.3 mg/L of manganese chloride, 1-4 mg/L of nickel chloride, 0.3-0.8 mg/L of indoleacetic acid and pH 7.5-8.0;

step 3: centrifuging at 6000 rpm for 10 min, collecting thallus, re-suspending thallus with sterile seawater, and regulating thallus concentration with Maillard turbidimetry.

The clostridium butyricum is obtained from China center for industry microbiological culture collection center, with a preservation number of CICC23847, and is formed by performing expansion culture according to the following steps:

step 1: freeze-dried powder of clostridium butyricum 0.1. 0.1 g (viable count 0.5X10) ¹⁰ ~10 ¹⁰ cfu/g) is fully dissolved in 1 ml sterile distilled water, strains are streaked and activated on a reinforced clostridium agar medium (RCM), and the culture is carried out for 1 to 2 days in an anaerobic incubator at 37 ℃, so that round single colonies appear on a flat plate; the reinforced clostridium agarThe fat culture medium comprises 10 g/L of peptone, 10 g/L of beef powder, 3 g/L of yeast powder, 5 g/L of glucose, 1 g/L of soluble starch, 5 g/L of sodium chloride, 3 g/L of sodium acetate, 0.5 g/L of cysteine hydrochloride, 0.15 g/L of manganese sulfate, 1.5 g/L of dipotassium hydrogen phosphate, 0.25 g/L of calcium chloride, 0.65 g/L of magnesium sulfate and 15 g/L of agar, and has pH value of 6.8-7.2;

step 2: selecting single bacterial colony, inoculating the bacterial colony into a reinforced clostridium liquid culture medium, standing the bacterial colony in an anaerobic incubator at 37 ℃ for anaerobic culture for 1-2 days, and culturing until the bacterial strain grows to a logarithmic phase; the reinforced clostridium liquid culture medium comprises 10 g/L peptone, 10 g/L beef powder, 3 g/L yeast powder, 5 g/L glucose, 1 g/L soluble starch, 5 g/L sodium chloride, 3 g/L sodium acetate, 0.5 g/L cysteine hydrochloride, 0.15 g/L manganese sulfate, 1.5 g/L dipotassium hydrogen phosphate, 0.25 g/L calcium chloride and 0.65 g/L magnesium sulfate, and has a pH value of 6.8-7.2;

step 3: centrifuging at 6000 rpm for 10 min, collecting thallus, re-suspending thallus with sterile seawater, and regulating thallus concentration with Maillard turbidimetry.

Example 2:

rhodopseudomonas palustris, clostridium butyricum, oak leaf extract and aloe leaf extract were prepared in substantially the same manner as in example 1. Except that the culture temperature of rhodopseudomonas palustris is 35 ℃ and the illumination intensity is 3000 lux. The volume fraction of ethanol leaching solution of oak leaf extract and aloe leaf extract is 85%, the feed-liquid ratio is 0.1 g/mL, and the leaching time is 2.5 h. Rhodopseudomonas palustris 10 is added into each gram of feed ⁷ cfu, clostridium butyricum 10 ⁵ cfu, oak leaf extract 2 mg, aloe leaf extract 8 mg;

example 3:

rhodopseudomonas palustris, clostridium butyricum, oak leaf extract and aloe leaf extract were prepared in substantially the same manner as in example 1. Except that the culture temperature of rhodopseudomonas palustris is 33 ℃ and the illumination intensity is 2500 lux. The volume fraction of ethanol leaching solution of oak leaf extract and aloe leaf extract is 75%, the feed-liquid ratio is 0.07 g/mL, and the leaching time is 2 h. Rhodopseudomonas palustris 10 is added into each gram of feed ⁶ cfu, clostridium butyricum 10 ⁶ cfu, oak leaf extraction1.5. 1.5mg, aloe vera leaf extract 6mg;

in the example 3, the colony morphology of rhodopseudomonas palustris and clostridium butyricum is shown in the figure 1, rhodopseudomonas palustris (A) is a brown circular colony, the edge of the colony is neat and smooth, and the diameter of the colony is 0.3 mm-0.60 mm; clostridium butyricum (B) is a milky white circular colony, the surface of the colony is smooth and provided with bulges, the edges are neat, and the diameter of the colony is 1-3 mm.

Experiment:

in the following experiments, the stichopus japonicus is taken from the breeding center of the sea water science institute of Liaoning province, the stichopus japonicus monomer mass is 3.17+/-0.41 and g, and is temporarily cultivated in four aquariums (100 cm multiplied by 80 cm multiplied by 75 cm) with the same specification, and 90 stichopus japonicus is planted in each box. The aquariums are respectively numbered A, B, C, D. After the stichopus japonicus is temporarily raised for one week, feeding feed according to 5% of the stichopus japonicus body mass, feeding feeds added with the compound micro-ecological preparation of the embodiment 1-3 of the invention into the corresponding aquarium of the embodiment 1, 2 and 3 of the embodiment A, B, C respectively, and feeding feeds without adding the compound micro-ecological preparation into the aquarium of the embodiment D of the invention into the corresponding aquarium of the embodiment D of the invention.

The feeding experiment was continued for 60 days with 3 groups of replicates, 3 replicates per treatment. Feeding the seawater for 1 time every day, wherein the feeding time is 15:00 pm, the aeration is kept during the feeding period, the water is changed every 2 days, the seawater temperature is 16-18 ℃, the pH is 7.5-8.3, and the salinity is 30. The dissolved oxygen is not lower than 5 mg/L, and the ammonia nitrogen is not higher than 0.5 mg/L. After the 60-day feeding experiment is finished, the stichopus japonicus is subjected to a toxicity attack experiment by using vibrio splendidus, vibrio harveyi and vibrio parahaemolyticus respectively. Vibrio splendidus (MCCC 1A 04096), vibrio harveyi (MCCC 1A 00232) and Vibrio parahaemolyticus (MCCC 1A 16298) are all purchased from the marine microorganism strain collection management center.

The toxicity attack experiment adopts a dipping method, 25 stichopus japonicus are taken in each group, 3 groups of parallel are arranged, and the dipping concentration of pathogenic vibrio is 1 multiplied by 10 ⁸ cfu/mL. During the period of toxin attacking, the feed is normally fed without changing water, and the residual feed and the excrement are removed by siphoning every day. The vibrio morbidity and the cumulative mortality of stichopus japonicus within 15 days after the challenge are recorded. Before and after the start of the toxicity attack experiment, 3 stichopus japonicus selenka are randomly selected from each water tank, and the abdominal cavity is dissected to obtain body cavity liquid. Mixing the cavity liquids of radix Morinae Bulleyanae, centrifuging at 4deg.C and 3500 rpm for 1For 0 min, the supernatant was used to determine the immunoenzymatic activity. The immune enzyme activity of the stichopus japonicus is measured by adopting a test box of Nanjing established technology limited company, and the specific measuring steps are carried out according to the specification of the kit. The enzyme activity detection experiment is repeated for 3 times, and the enzyme activity is expressed in terms of specific activity and the unit is U/mg protein. After the toxicity attack experiment is finished, 3 stichopus japonicus and 3 water samples are randomly taken from each group, intestinal tissues are obtained by dissecting the stichopus japonicus, the intestinal tissues are homogenized for 10 min by using 30 mLPBS buffer solution, and the homogenates are combined. Diluting intestinal tissue homogenate and a culture water sample by 10 times respectively with sterile physiological saline, respectively taking 0.05 ml diluted homogenate and the culture water sample, coating the diluted homogenate and the culture water sample on a TCBS vibrio selective culture medium, culturing the culture medium in an inverted manner at 28 ℃, and counting the number of the intestinal tracts of the stichopus japonicus and the vibrio in a culture water body after 24 h.

The effect of the complex microecological preparation on the immune enzyme activity of the apostichopus japonicus is shown in Table 1, and among the selected enzyme activity indexes, acid phosphatase (ACP) and alkaline phosphatase (AKP) are important hydrolytic enzymes for the cell internal parameters and immune defense activities of the coelom of the apostichopus japonicus. Superoxide dismutase (SOD) is an important antioxidant enzyme, and can remove excessive oxygen free radicals in the organism and repair damaged cells. Lysozyme (LSZ) cleaves glycosidic linkages in bacterial cell wall peptidoglycans, causing cell lysis and eliminating pathogenic bacteria invading the organism.

TABLE 1 Effect of composite microecological preparation on the immune enzyme Activity of offensive Apostichopus japonicus

From table 1, before the toxicity attack experiment, the immune enzyme indexes of the stichopus japonicus in the examples 1-3 are obviously higher than those of the control example, which indicates that the immune function of the stichopus japonicus is improved in the feeding stage, and the probiotic effect of the compound microecological preparation is reflected. After the toxicity attack experiment is finished, the immune index of the stichopus japonicus of the control example is obviously reduced, which indicates that the immunity enzyme activity of the stichopus japonicus is inhibited by pathogenic vibrio infection and the immune function is destroyed. The immunity indexes of the stichopus japonicus in each embodiment group are not greatly different from those before toxicity attack, so that the compound microecological preparation can improve the phosphatase response capability of the stichopus japonicus, effectively inhibit oxidative damage, promote the stimulus of the stichopus japonicus to the pathogenic bacteria and improve the immunity function of the stichopus japonicus.

The effect of each experimental group on the cumulative mortality of Vibrio vulnificus, vibrio harveyi, vibrio parahaemolyticus stichopus japonicus is shown in FIG. 2, FIG. 3 and FIG. 4. The results show that: the first dead individuals after the different pathogenic vibrios attack the viruses are all found in the control, and the cumulative mortality of stichopus japonicus in each example is obviously lower than that of the control. Wherein, after the Vibrio splendidus and Vibrio harveyi attack the toxin, the cumulative mortality rate of stichopus japonicus in example 3 is lower than that of other examples. After the challenge of Vibrio parahaemolyticus, the cumulative mortality rate of stichopus japonicus in example 1 is lower than that of other examples.

The effect of each experimental group on the disease condition of the toxin-attacking stichopus japonicus is shown in fig. 5, and the stichopus japonicus of the control example and each example has typical symptoms of 'putrescence Pi Zeng syndrome' with different degrees, including body atrophy, head shaking, dirt removal, skin formation and the like. The body surface ulcer area of the stichopus japonicus in each embodiment is smaller than that of the control, and the response capability, the adhesion capability and the relative immune protection rate to external stimulus are higher than those of the control.

The inhibiting effect of each experimental group on vibrio splendidus, vibrio harveyi and vibrio parahaemolyticus is shown in fig. 6, 7 and 8, and the number of vibrio splendidus, vibrio harveyi and vibrio parahaemolyticus in intestinal tracts and culture water bodies of stichopus japonicus in each embodiment is obviously smaller than that of a control example. Wherein, the compound microecological preparation in the embodiment 1 has higher inhibition effect on vibrio parahaemolyticus than other embodiments, and the compound microecological preparation in the embodiment 3 has higher inhibition effect on vibrio harveyi than other embodiments.

The experimental results show that the invention has small dosage, the components cooperate and are synergistic with each other, the invasion and the infection of various pathogenic vibrios can be resisted in a broad spectrum, the proliferation of pathogenic bacteria in intestinal tracts of stichopus japonicus and culture water bodies can be effectively inhibited, and the immune function and the disease resistance of the stichopus japonicus are obviously enhanced.

Claims (4)

1. A composite microecological preparation for inhibiting pathogenic vibrio for culturing stichopus japonicus is characterized by comprising rhodopseudomonas palustrisRhodopseudomonas palustris) Clostridium butyricumClostridium butyricum) Mixing oak leaf extract and aloe leaf extract to obtain feed additive, wherein the additive amount of each gram of feed is rhodopseudomonas palustris 10 ⁶ cfu, clostridium butyricum 10 ⁶ cfu, oak leaf extract 1.5mg, aloe leaf extract 6mg; the oak leaf extract and the aloe leaf extract are prepared according to the following method: respectively cleaning oak leaf and aloe leaf with distilled water, and drying in shade at 25deg.C; homogenizing and crushing oak leaves and aloe leaves by a homogenizer respectively, and adding an ethanol solution with the volume fraction of 65-85%, wherein the feed-liquid ratio is 0.05-0.1 g/mL; leaching for 1-2.5 h at normal temperature under the action of ultrasonic waves with the working frequency of 40 KHZ and the power of 80W; loading the leaching solution into ultrafiltration membrane device, filtering under 0.06Mpa to remove impurities, concentrating under reduced pressure, and lyophilizing to obtain lyophilized powder, namely oak leaf extract and aloe leaf extract; the rhodopseudomonas palustris is purchased from China center for industrial microbiological culture collection, the preservation number is CICC23812, and the clostridium butyricum is from the China center for industrial microbiological culture collection, and the preservation number is CICC23847.

2. The complex microecological preparation for inhibiting pathogenic vibrio for stichopus japonicus according to claim 1, wherein the rhodopseudomonas palustris is prepared by the following steps:

step 1: the viable count of 0.1 g is 0.5X10 ¹⁰ ~10 ¹⁰ cfu/g rhodopseudomonas palustris freeze-dried powder microbial inoculum is dissolved in 1 ml sterile distilled water, and strains are streaked and activated on a solid culture medium, wherein the solid culture medium comprises 15.0 g/L tryptone, 5.0 g/L soyase, 5.0 g/L sodium chloride and 20 g/L agar, and pH 7.5-8.0; culturing in an anaerobic incubator at the illuminance of 2000-3000 lux and the temperature of 30-35 ℃ until red single colonies appear on a plate;

step 2: selecting red single bacterial colony, inoculating the red single bacterial colony into a rhodopseudomonas palustris liquid culture medium, wherein the culture temperature is 30-35 ℃, the illumination intensity is 2000-3000 lux, and carrying out stationary culture in an anaerobic incubator for 3-5 days, wherein the liquid culture medium comprises 15.0 g/L of tryptone, 5.0 g/L of soyase and 5.0 g/L of sodium chloride, and pH 7.5-8.0;

step 3: the cells were collected by centrifugation at 6000 rpm for 10 min and resuspended in sterile seawater.

3. The composite microecological preparation for inhibiting pathogenic vibrio for stichopus japonicus according to claim 2, wherein the solid medium and the liquid medium are further added with the following components: 0.1-0.3 g/L of magnesium chloride, 2-5 mg/L of ferrous sulfate, 0.5-1.5 mg/L of zinc sulfate, 0.1-0.3 mg/L of manganese chloride, 1-4 mg/L of nickel chloride and 0.3-0.8 mg/L of indoleacetic acid.

4. The complex microecological preparation for inhibiting pathogenic vibrio for stichopus japonicus according to claim 3, wherein the clostridium butyricum is prepared by the following steps:

step 1: the viable count of 0.1 g is 0.5X10 ¹⁰ ~10 ¹⁰ Fully dissolving cfu/g clostridium butyricum freeze-dried powder microbial inoculum in 1 ml sterile distilled water, streaking and activating strains on a reinforced clostridium agar culture medium, and culturing in an anaerobic incubator at 37 ℃ until a circular single colony appears on a flat plate; the reinforced clostridium agar medium comprises 10 g/L peptone, 10 g/L beef powder, 3 g/L yeast powder, 5 g/L glucose, 1 g/L soluble starch, 5 g/L sodium chloride, 3 g/L sodium acetate, 0.5 g/L cysteine hydrochloride, 0.15 g/L manganese sulfate, 1.5 g/L dipotassium hydrogen phosphate, 0.25 g/L calcium chloride, 0.65 g/L magnesium sulfate and 15 g/L agar, and has pH value of 6.8-7.2;

step 2: selecting single bacterial colony, inoculating the single bacterial colony into a reinforced clostridium liquid culture medium, and standing the single bacterial colony in an anaerobic incubator for anaerobic culture at 37 ℃ for 1-2 days; the reinforced clostridium liquid culture medium comprises 10 g/L peptone, 10 g/L beef powder, 3 g/L yeast powder, 5 g/L glucose, 1 g/L soluble starch, 5 g/L sodium chloride, 3 g/L sodium acetate, 0.5 g/L cysteine hydrochloride, 0.15 g/L manganese sulfate, 1.5 g/L dipotassium hydrogen phosphate, 0.25 g/L calcium chloride and 0.65 g/L magnesium sulfate, and has a pH value of 6.8-7.2;

step 3: the cells were collected by centrifugation at 6000 rpm for 10 min and resuspended in sterile seawater.

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