CN111944715B - A strain of Shewanella putrefaciens and its application, aquafeed and aquaculture method - Google Patents

Abstract

The invention discloses Shewanella putrefaciens and application thereof, an aquatic feed and an aquaculture method, wherein the Shewanella putrefaciens is Shewanella putrefeaciens Z9 and is preserved in China center for type culture collection with the preservation number: CCTCC M2020210, and the preservation date is 6 months and 16 days in 2020. The Shewanella putrefaciens is high in safety, and has the functions of improving the utilization rate of plant protein of shrimp and crab production animals, promoting the growth of shrimps and crabs, improving the damage of intestinal peritrophic membranes of shrimps and crabs, improving the disease resistance and the like.

Description

A strain of Shewanella putrefaciens and its application, aquafeed and aquaculture method

technical field

The invention relates to the field of aquaculture, in particular to a strain of Shewanella putrefaciens and its application, aquafeed and aquaculture method.

Background technique

With the increase of aquaculture production in our country year by year, the shortage of fishmeal resources is becoming more and more serious. The use of plant protein to replace fishmeal is a common strategy in the feed industry. However, the anti-nutritional factors in plant raw materials are one of the important factors that limit the application of plant protein in aquaculture compound feed. If it is found that soybean globulin can easily cause intestinal oxidative damage of aquatic animals such as grass carp, Jian carp and Chinese mitten crab, especially easy to cause damage to the peritrophic membrane structure of the hindgut of Chinese mitten crab, reduce the activity of digestive enzymes, and destroy the intestinal microecology Balance, and ultimately inhibit the growth performance of Eriocheir sinensis.

Furthermore, animal gut microbes are key factors affecting host metabolism, immune status and body health. Due to the plasticity of intestinal microbial structure, it is possible to optimize the structure of animal intestinal microbial flora by artificially supplementing a sufficient amount of probiotics, promote animal digestion of feed, nutrient absorption and energy supply, promote animal growth, and enhance host health. Immune defense and protection of the host from pathogenic infection, etc.

At present, most of the relevant research on the screening of probiotics in aquatic animals focuses on Bacillus, lactic acid bacteria and other strains, and the focus is on antagonizing pathogenic bacteria and improving animal immunity. Therefore, how to screen out the corresponding strains to make them It is an urgent problem to be solved to improve the utilization efficiency of feed raw materials and reduce the damage caused by the anti-nutritional effect of soybean antigen protein to the intestinal tract of aquatic animals.

Contents of the invention

Aiming at the above-mentioned technical problems, the present invention provides a strain of Shewanella putrefaciens and its application, aquatic feed and aquaculture method. The said Shewanella putrefaciens has high safety and has the ability to improve the resistance of shrimp and crab production animals to vegetable protein. Utilization rate, promoting the growth of shrimp and crabs, improving the damage of intestinal peritrophic membrane of shrimps and crabs, and improving disease resistance.

In order to achieve the above object, the present invention adopts the following technical solutions:

On the one hand, a strain of Shewanella putrefaciens is provided, said Shewanella putrefaciens is Shewanella putrefaciens (Shewanella putrefaciens) Z9, and it is preserved in the China Type Culture Collection Center, the address is Wuchang District, Wuhan City, Hubei Province Wuhan University, the deposit number is: CCTCC M 2020210, and the deposit date is June 16, 2020.

Also provided is an application of the above-mentioned Shewanella putrefaciens Z9 in the preparation of antibacterial preparations.

Also provided is an application of the above-mentioned Shewanella putrefaciens Z9 in promoting the growth of crabs.

Also provided is an application of the above-mentioned Shewanella putrefaciens Z9 in improving the digestibility of crabs.

Preferably, the application includes increasing the activity of protease and/or lipase in crab intestinal tract.

Also provided is an application of the above-mentioned Shewanella putrefaciens Z9 in improving intestinal health of crabs.

Preferably, the application includes reducing serum diamine oxidase and intestinal tumor necrosis factor-α levels of crabs, and/or improving intestinal tissue morphology of crabs.

Also provided is an application of the above-mentioned Shewanella putrefaciens Z9 in promoting the metabolic activity of intestinal microorganisms of crabs.

Also provided is an application of the above-mentioned Shewanella putrefaciens Z9 in improving disease resistance of crabs.

Also provided is a crab breeding feed, which includes the above-mentioned Shewanella putrefaciens Z9.

Preferably, in the crab breeding feed, the concentration of the Shewanella putrefaciens Z9 is 10 ⁷ -10 ⁹ CFU/g.

Also provided is a crab breeding method, which uses the above-mentioned aquatic feed to feed the crabs 1-3 times a day, and feeds 4% of the body weight each time, and the breeding time is 5-10 weeks.

The present invention at least has the following beneficial effects:

The Shewanella putrefaciens Z9 in the present invention can be added to the shrimp and crab breeding feed, by improving the utilization efficiency of the soybean globin in the shrimp and crab, improving the metabolic activity of the intestinal microorganisms of the shrimp and crab, and improving the activity of digestive enzymes at the same time to promote growth; further, the Shewanella putrefaciens Z9 can also maintain the peritrophic membrane morphology of shrimp and crab intestines, repair the intestinal microecology, and improve the intestinal health of shrimp and crabs. In addition, the Shewanella putrefaciens Z9 also has antibacterial properties, and can improve the disease resistance of shrimp and crabs by protecting the body's immune system.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present invention. Ordinary technicians can also obtain other drawings based on these drawings on the premise of not paying creative work.

Fig. 1 is the colony morphology of Shewanella putrefaciens Z9 of the present invention;

Fig. 2 is the growth status and the hemolysis status of Shewanella putrefaciens Z9 of the present invention on the glycinin medium;

Fig. 3 is the antagonistic effect of Shewanella putrefaciens Z9 of the present invention on Vibrio anguillarum, Staphylococcus aureus and Aeromonas hydrophila respectively;

Fig. 4 is the tolerance ability produced by Shewanella putrefaciens Z9 of the present invention to artificial simulated crab gastric juice (A) and intestinal juice (B);

Fig. 5 is the hydrophobicity of Shewanella putrefaciens Z9 of the present invention in three kinds of hydrocarbons (xylene, chloroform and ethyl acetate) respectively;

Fig. 6 is the impact of Shewanella putrefaciens Z9 of the present invention on the survival rate (a) and weight gain rate (b) of Chinese mitten crab;

Fig. 7 is the effect of Shewanella putrefaciens Z9 of the present invention on the activity of amylase (A), trypsin (B) and lipase (C) in the hepatopancreas and intestinal tract of Chinese mitten crab young crabs;

The influence of Shewanella putrefaciens Z9 of the present invention on Chinese mitten crab serum diamine oxidase (a) and intestinal TNF-α of Fig. 8;

The influence of Fig. 9 Shewanella putrefaciens Z9 of the present invention on the intestinal histological morphology of Chinese mitten crab;

Fig. 10 shows the effect of Shewanella putrefaciens Z9 of the present invention on the aerobic metabolism of cultivable microorganisms in the intestinal tract of Chinese mitten crab. (A) AWCD activity; (B) relative utilization of four carbon sources;

Fig. 11 shows the effect of Shewanella putrefaciens Z9 of the present invention on the survival rate of Eriocheir sinensis challenged with Aeromonas hydrophila.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Example 1:

This example provides a strain of Shewanella putrefaciens (Shewanella putrefaciens) Z9 (hereinafter referred to as "Z9"), which is preserved in the China Center for Type Culture Collection, the preservation number is: CCTCC M 2020210, and the preservation date is: 2020 June 16.

The above strain information is as follows:

1. Source of the strain

The strain was isolated from the intestinal tract of Chinese mitten crab (Eriocheirsinensis) fed a diet containing glycinin for 8 weeks.

2. Morphological features

Gram staining was negative. As shown in Figure 1, the colony cultured upside down on the beef extract-peptone medium plate at 28°C for 12 hours was light yellow and round with a smooth surface, and the center of the colony was darker and orange.

3. Utilize the ability of soybean globulin

A culture plate was prepared by using a solid medium added with glycinin (mass fraction 1%), and several holes (diameter 6 mm) were drilled on the medium with a sterile drill, and then 60 μL of the Z9 suspension was added to each plate on average. After opening the wells, the culture plate was incubated at 28°C for 24 hours to identify the ability of Z9 to decompose and utilize glycinin.

The results are shown in Figure 2(a). Since the agar plate added with glycinin was milky white, after adding Z9 suspension for culture, a transparent area S1 appeared around the opening, which means that Z9 has the ability to decompose and utilize glycinin. .

4. Enzyme production characteristics

1) Dip a single colony of Z9 with a toothpick, inoculate it on an agar plate containing soluble starch (1% mass fraction), incubate upside down at 28°C for 12 hours, and then evenly spray 1% Lugol’s iodine solution on the plate Finally, let it stand for 3-5 minutes, observe whether there is a transparent zone around the Z9 bacteria, and identify whether the strain can secrete amylase.

2) Use a toothpick to pick up a single colony of Z9, inoculate it on an agar plate containing skimmed milk (mass fraction 2%), culture it upside down at 28°C for 12 hours, observe whether there is a transparent area around the Z9 bacteria, and identify whether the strain can secrete protease .

3) Use a toothpick to pick up a single colony of Z9, inoculate it on an agar plate containing triacylglycerol and rhodamine B, culture it upside down at 28°C for 12 hours, and observe whether there is an orange fluorescent halo around the bacterial colony under ultraviolet light To identify whether the strain can secrete lipase.

The enzyme production ability of Z9 is expressed by the ratio of the diameter of the transparent area/orange fluorescent halo and the diameter of the colony. The results are shown in Table 1.

Table 1 Enzyme-producing ability of Shewanella putrefaciens Z9

Note: All activity determinations were repeated three times, and the enzyme activity was expressed by the ratio of the transparent area/orange fluorescent halo diameter to the colony diameter. + represents 1.5-2.0, ++ represents 2.0-2.5, +++ represents 2.5-3.0, - represents no enzyme activity.

As shown in Table 1, Z9 was able to secrete protease and lipase, but not amylase.

5. Pathogenicity

Take the above-mentioned Z9 frozen bacteria and Aeromonas hydrophila, and inoculate them into beef extract-peptone liquid medium at a ratio of 1:1000 to recover, shake the bacteria at 28°C and 220rpm/min for 12h, and use an inoculation loop Dip the above-mentioned Aeromonas hydrophila bacteria solution, streak on the beef extract-peptone medium plate, incubate upside down at 28°C for 12 hours, dip a single colony of Z9 with a toothpick, spot it on the blood plate, and incubate upside down at 28°C for 12h.

The results are shown in Figure 2(b), Z9 (i.e. ① and ②) has no hemolysis phenomenon on the blood plate, but the pathogenic bacteria Aeromonas hydrophila (i.e. ③ and ④) has hemolysis circles on the blood plate , which shows that Z9 has no obvious pathogenicity and is relatively safe.

6. In vitro antibacterial properties

Three common pathogens in aquaculture: Vibrio anguillarum (ATCC 43308), Aeromonas hydrophila (ATCC 35654) and Staphylococcus aureus (ATCC 25923) were used as indicator strains to detect the antibacterial activity of Z9.

Specifically, after culturing the three indicator pathogenic bacteria for 12 hours, adjust the concentration of the bacteria with PBS to about OD ₆₀₀ of about 0.8, absorb 100 μL respectively and apply it on a fresh beef extract-peptone plate, and drill several holes on the medium (diameter 6mm), and then 60 μL of Z9 suspension was added to each hole on average, and the culture plate was incubated at 28°C for 24 hours to observe whether there was an inhibition zone around the colony, and measure the diameter of the inhibition zone to indicate Antipathogenic ability of Z9 strain.

It can be observed from Figure 3 that there are obvious antibacterial transparent circles around the colonies of Vibrio anguillarum, Staphylococcus aureus and Aeromonas hydrophila, which shows that Z9 can significantly Inhibits the growth of Vibrio anguillarum and Staphylococcus aureus.

7. Artificial gastrointestinal fluid tolerance

Prepare simulated gastric juice by mixing pepsin (0.3 mg/mL) and NaCl (mass fraction 0.5%) in PBS (adjusted to pH 3), then pass through a 0.22 μm filter to sterilize, and then resuspend in simulated gastric juice Z9 bacteria were precipitated and incubated at 28°C for 2h. Count the live bacteria on the plate at three time points of 0h, 1h and 2h, and calculate the survival rate of Z9 respectively.

And add trypsin (0.1mg/mL) and ox bile salt (mass fraction 0.3%) to PBS, then adjust the pH to 6.8 to prepare simulated intestinal juice, then pass through a 0.22μm filter membrane to sterilize, and then resuspend with simulated intestinal juice Z9 bacteria were precipitated and incubated at 28°C for 4h. Count the live bacteria on the plate at three time points of 0h, 2h, and 4h, and calculate the survival rate of Z9 respectively.

It can be observed from Figure 4 that Z9 has a strong tolerance to simulated gastric juice and simulated intestinal juice, and its survival rate is about 80% when it is in simulated gastric juice for 2 hours, and the survival rate is close to 80% when it is in simulated intestinal juice for 4 hours Left and right, which shows that it can play its role in the animal intestine for a long time.

8. Cell surface hydrophobicity

The overnight cultured Z9 bacterial pellet was washed twice with PBS, resuspended, and the absorbance of the cell suspension was measured at 600 nm (A ₀ ). Then take three parts of the cell suspension, respectively add equal volumes of hydrocarbons: xylene, chloroform and ethyl acetate, and vortex for 2 minutes.

The two phases were separated for 1 hour, and the aqueous phase was gently removed to measure its absorbance (A _t ) at 600 nm, and the cell surface hydrophobicity was further calculated according to the following formula:

Hydrophobic rate (%) = (A ₀ -A _t )/A ₀ ×100

Among them, A ₀ represents the absorbance before mixing with hydrocarbons, and A _t represents the absorbance after mixing with hydrocarbons for 1 hour.

It can be seen from Figure 5 that Z9 has strong hydrophobicity among the three hydrocarbons, indicating that Z9 has a high aggregation ability, and thus can play a concentrated role in improving the intestinal health and disease resistance of aquatic animals.

Example 2:

The present embodiment provides the separation method of Shewanella putrefaciens Z9 (hereinafter referred to as "Z9") described in embodiment 1, which comprises the steps:

S1. According to tryptone 5g/L, beef extract 5g/L, soybean globulin 20g/L, yeast extract 2g/L, KH ₂ PO ₄ 2g/L, MgSO ₄ 7H ₂ O 2g/L, NaCl 5g/L L, the ratio of agarose 20g/L to prepare glycinin medium, after autoclaving, pour the plate in the ultra-clean bench to obtain a solid plate of glycinin medium;

S2. Mix the intestinal contents of crabs (such as Chinese mitten crab) with PBS buffer at a ratio of 1:9 (w/v, g/ml), centrifuge at 1500 rpm/min, and take 100 μL of supernatant Apply to the glycinin medium solid plate;

S3. After upside-down culture at 28°C for 24 hours, colonies were screened according to the size, shape, and color of the colonies, and the slant planes of the screened colonies were stored and numbered. At the same time, the screened strains were subjected to physiological, biochemical and 16S rRNA molecular identification. To obtain the Shewanella putrefaciens Z9.

Example 3:

The present embodiment provides a kind of method that adopts Shewanella putrefaciens Z9 (hereinafter referred to as "Z9") described in Example 1 to carry out crab breeding, which specifically includes the following steps:

S1. Temporary raising of crab seedlings: buy 1,000 crab seedlings (such as Chinese mitten crab seedlings), and all crab seedlings will be temporarily raised for 2 weeks before the formal breeding experiment. Then the crab seedlings (0.28 ± 0.01g) with complete appendages are randomly divided into 25 300L culture buckets;

There are 5 treatments in total: normal group (C), glycinin group (11S), probiotic group 1 (SP1), probiotic group 2 (SP3), probiotic group 3 (SP 3), 5 for each treatment Parallel, 40 crab seedlings in each parallel;

S2. Feed preparation: Feed preparation was carried out according to the feed raw material formula table shown in Table 2; wherein, the feed of group 11S used 8% soybean globulin in mass fraction to replace casein at the same protein level; fish oil and sodium alginate were retained during feed preparation, Save it for subsequent spraying; then use a twin-screw extruder to make the feed into pellets with a diameter of 1.5 mm, and air-dry until the moisture content is less than 10%, and store it at -20°C for later use;

Add the strain of Shewanella putrefaciens Z9 into beef extract-peptone medium, culture at 28°C for 18 hours with shaking at 180rpm/min to obtain a bacterial suspension; then centrifuge the bacterial suspension at 5000×g for 20min at 4°C to collect For Z9 cells, add PBS to resuspend the Z9 cells, and adjust the final concentration of Z9 cells to 2.0×10 ⁸ , 2.0×10 ⁹ and 2.0×10 ¹⁰ CFU/mL to obtain three concentrations of Z9 bacterial suspensions;

Then take 10mL of Z9 bacterial suspensions of three concentrations to homogenize thoroughly, and spray them on 100g of 11S group feed pellets respectively, so as to obtain corresponding Z9 concentrations of 10 ⁷ CFU/g, 10 ⁸ CFU/g, and 10 ⁹ CFU /g three different concentrations of probiotic feed (that is, crab breeding feed, respectively corresponding to feeding probiotic group 1 (SP1), probiotic group 2 (SP 3), probiotic group 3 (SP 3) group crab seedlings ); meanwhile, get 100g control group and remaining 11S group feed respectively and spray 10mL of PBS (not containing probiotics);

After the 5 groups of experimental feeds were air-dried in a cool place, fish oil and sodium alginate were mixed according to the ratio of the feed formula, and the mixture was evenly sprayed on the surface of the above 5 groups of feeds, and then stored at 4°C for later use;

Sampling and testing the effective concentration of bacteria in the feed every day, when the concentration is lower than the preset concentration of the experiment, a new batch of probiotic feed will be prepared.

Table 2 Feed Raw Material Formula Table (g)

Among them: ^aShanghai Sangon Bioengineering Co., Ltd.

^B -complex vitamins (per 100 grams): Retinyl acetate, 0.043 grams; Vitamin B1 hydrochloride, 0.15 grams; Vitamin B2, 0.0625 grams; Calcium pantothenate, 0.3 grams; Hydrochloric acid, 0.3 grams; Vitamin B6 hydrochloride , 0.225 grams; p-aminobenzoic acid, 0.1 grams; vitamin C, 0.5 grams; vitamin H, 0.005 grams; folic acid, 0.025 grams; vitamin D3, 0.0075 grams; vitamin E, 0.5 grams; vitamin K, 0.05 grams; Alcohol, 1 g; all vitamins supplemented by cellulose to 100 g.

^cMultiple minerals (per 100 grams): Potassium dihydrogen phosphate, 21.5 grams; Sodium dihydrogen phosphate, 10 grams; Calcium dihydrogen phosphate, 26.5 grams; Calcium carbonate, 10.5 grams; Potassium chloride, 2.8 grams; Heptahydrate sulfuric acid Magnesium, 10 g; Aluminum Chloride Hexahydrate, 0.024 g; Zinc Sulphate Hexahydrate, 0.476 g; Manganese Sulphate Hexahydrate, 0.143 g; Potassium Iodide, 0.023 g; Copper Chloride Dihydrate, 0.015 g; 0.14 g; Calcium lactate, 16.5 g; Ferric citrate, 1 g; All components filled with cellulose to 100 g;

S4, throwing something and feeding: 5 groups of experimental feeds are fed respectively to 5 groups of crab seedlings, fed 1-3 times every day, and fed by 4% of body weight every day; starved for 24h after breeding for 5-10 weeks (preferably 8 weeks), 10 young crabs were taken from each group for weighing, blood lymph and tissue sample collection.

The efficacy of the Shewanella putrefaciens Z9 was assessed as follows:

(1) The role of Z9 in promoting the growth performance of crab seedlings

After breeding, weigh the total weight of crab seedlings in each group, and calculate the survival rate and weight gain rate of each group.

Among them, survival rate (%)=final number/initial number, weight gain rate (%)=(final weight-initial weight)×100/initial weight.

As shown in Figure 6(a), compared with group C, the survival rate of 11S group was significantly lower, but the survival rate of SP group was significantly higher than that of 11S group, and there was no difference from group C. At the same time, as shown in Figure 6(b), the weight gain rate of the SP group was significantly improved compared with that of the 11S group, which shows that the Shewanella putrefaciens Z9 in the present invention can significantly improve the survival rate of juvenile crabs and rapidly promote the weight growth of juvenile crabs.

Note: "*" in Figure 6 represents the difference between the two groups, and *: P<0.05, **: P<0.01, and the data of SP group in (a)-(b) are all SP1, SP2 and SP3 The mean of the group data.

(2) Shewanella putrefaciens Z9 improves the digestive ability of juvenile crabs

The hepatopancreas and intestinal tissues of Eriocheir sinensis were collected separately, and the amylase, protease and fat in the hepatopancreas and intestinal tract of juvenile crabs were measured using Nanjing Jiancheng kits (Cat.No. C016-1, A080-2 and A054-1). For enzyme activity, the operation steps were performed according to the instructions of the kit.

From the results in Figure 7, it can be seen that compared with group C, the activity of trypsin in the hepatopancreas and intestine of group 11S was significantly reduced, while the activity of trypsin in the intestine of young crabs was significantly improved in group SP. In addition, the intestinal lipase activity of juvenile crabs in SP group was significantly higher than that in C and 11S groups.

As mentioned above, Shewanella putrefaciens Z9 has a high ability to secrete protease and lipase, which helps to improve the activity of these two digestive enzymes in the intestinal tract of Chinese mitten crabs during the actual breeding process, and further improves the juvenile crab's ability to The utilization rate of the soybean globin makes the juvenile crab grow well, and at the same time can reduce the cost and increase the economic benefit.

Note: "*" in Figure 7 represents the difference between the two groups, and *: P<0.05, **: P<0.01, and the data of SP group in A-B are the average value of the data of SP1, SP2 and SP3.

(3) Shewanella putrefaciens Z9 improves intestinal health of juvenile crabs

1) Collect hemolymph from juvenile crabs, collect serum, and use Nanjing Jiancheng kit (A088-1) to measure diamine oxidase (DAO) activity in serum, and the operation steps are performed according to the kit instructions. Intestinal tissues of juvenile crabs were collected, and the concentration of tumor necrosis factor-α (TNF-α) in the intestinal tract of juvenile crabs was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Jianglai Biological Co., Ltd.).

As shown in Figure 8(a), the serum DAO activity of the 11S group was significantly higher than that of the C and SP groups, while Figure 8(b) showed that the intestinal TNF-α level of the 11S group was significantly higher than that of the C and SP groups. It shows that adding Shewanella putrefaciens Z9 can reduce serum DAO and intestinal TNF-α levels of juvenile crabs.

When the intestinal mucosa is damaged, the DAO in the intestinal epithelial cells is released into the plasma. The damaged intestinal mucosa can easily cause the host to be infected by pathogens, which in turn causes mast cells to secrete a large amount of pro-inflammatory cytokines such as TNF-α. Therefore, the use of Shewanella putrefaciens Z9 can significantly improve intestinal inflammatory damage in crabs caused by anti-nutritional factors in plant protein, and improving intestinal health is conducive to maintaining its digestive function.

Note: "*" in Figure 8 represents the difference between the two groups, and *: P<0.05, **: P<0.01, and the data of SP group in A-B are the average value of the data of SP1, SP2 and SP3.

2) 5 crabs were randomly selected in each group, and 2-3 cm intestinal segments were taken from the same part of the intestinal tract of each crab, fixed in 4% paraformaldehyde solution, dehydrated in ethanol, washed in toluene, and then equilibrated in xylene , and finally embedded in paraffin to make a solid wax block. Then, the buried intestine was sectioned with a rotary microtome to a thickness of about 5 μm, stained with hematoxylin-eosin and observed on an Axioskop microscope (BX51, Olympus, Tokyo, Japan).

It can be seen from Figure 9 that the intestinal mucosal folds of juvenile crabs are composed of columnar epithelial tissue, and the folds are developed in group C, and the peritrophic membrane tightly covers the surface of intestinal folds. In the 11S group, there was a gap between the peritrophic membrane and intestinal folds, and the peritrophic membrane was damaged and fell off. In the SP group, the intestinal folds increased, and the peritrophic membrane covered the surface of the intestinal folds.

Therefore, by using Shewanella putrefaciens Z9, the intestinal morphology of crabs can be significantly improved, especially the peritrophic membrane structure unique to the digestive tract of crustaceans can be well protected, and the damage caused by plant proteins to crab intestines can be repaired. Mechanical and chemical damage.

(4) The role of Shewanella putrefaciens Z9 in promoting the metabolic activity of intestinal microorganisms in crabs

The metabolic activity of gut microbes was analyzed using Biolog ^™ EcoPlates (Hayward, CA, United States). Biolog EcoPlates contain 31 carbon sources, which can be divided into four types: sugars and their derivatives (SD), fatty acids and their derivatives (FAD), amino acids and their derivatives (AAD) and secondary metabolites (ISM)[ 384]. Briefly, the intestinal tract of Eriocheir sinensis was made into a 10% (w:v) homogenate by adding sterile 0.9% saline. The homogenate was centrifuged at 3500 rpm for 10 minutes at 4° C., and then the suspension was filtered with a nitrocellulose membrane filter (50 μm pore size). The clear supernatant was diluted 100-fold, and 150 μl was used to inoculate 96 microtiter wells of the Biolog plate. The inoculated plate was incubated at 28° C., and the absorbance value of each well was measured at 590 nm with a BIOLOG plate reader every 24 h, and continuously monitored for 7 days. All assays were performed in triplicate samples.

The average color rendering (AWCD) of Biolog™ EcoPlates has been used as an indicator of the aerobic metabolism of cultivable bacteria in research. AWCD was calculated using the relative utilization of the 31 carbon sources after incubation for 72 h. The calculation formula of AWCD is as follows:

AWCD=∑(Ci-R)/ _n

where C _i is the OD value of each well, R is the OD value of the control well, and n is the number of substrates (31 in EcoPlates).

As shown in Figure 10(a), the intestinal microbial metabolic activity of juvenile crabs from high to low was group C>SP group>11S group. 10(b) showed that, compared with group C, the metabolic activity of intestinal microbes in 11S group to amino acids and their derivatives was significantly reduced, while that in SP group was significantly higher than that in 11S group. The ability of gut microbes in the SP group to utilize fatty acids and their derivatives was also improved.

This shows that by adding Shewanella putrefaciens Z9 to the plant protein feed, the metabolic activity of crab intestinal microorganisms can be significantly improved, the utilization rate of plant protein by crabs can be improved, and the effect of antinutritional factors such as soybean globinin on crabs can be reduced. Injuries caused by the intestinal tract.

Note: "*" in Figure 10 represents the difference between the two groups, and *: P<0.05, **: P<0.01, and the data of SP group in A-B are the average value of the data of SP1, SP2 and SP3.

(5) Effect of Shewanella putrefaciens Z9 on improving disease resistance of crabs

Take Aeromonas hydrophila and inoculate it into LB liquid medium for recovery, shake culture at 28°C and 180rpm/min for 12 hours, take the recovered bacteria solution and inoculate it into LB liquid medium at a ratio of 1:100 for expansion culture, at 28°C, The culture was continued for 24 hours at 180 rpm/min, and then centrifuged at 4°C and 12000 rpm/min for 10 minutes to collect the bacteria, resuspended with PBS buffer, and adjusted the concentration to 10 ⁹ CFU/mL. Take 30 juvenile crabs (such as Chinese mitten crab) from each group for the challenge experiment, inject Z9 bacterial liquid at the amount of 10 ⁶ CFU/g at the appendage joints, observe and record the death situation for 7 days, and calculate the survival rate.

The results are shown in Figure 11, after 48 hours, the survival rates among the three groups began to differ. Seven days after the challenge, the 11S group had the lowest survival rate, and the SP group had the highest survival rate.

As mentioned above, Shewanella putrefaciens Z9 has strong inhibitory ability to common pathogenic bacteria such as Aeromonas hydrophila, Vibrio anguillarum and Staphylococcus aureus in the process of crab farming. This shows that Shewanella putrefaciens Z9 can effectively improve the infection ability of crabs against viruses and pathogens (such as Aeromonas hydrophila), enhance the comprehensive immunity and disease resistance of crabs, and further improve the survival rate.

In summary, the Shewanella putrefaciens Z9 isolated by the present invention can be added to crab feeds containing high plant protein as a safety functional feed additive, and Shewanella putrefaciens Z9 can significantly improve the crab feed caused by glycinin. Intestinal peritrophic membrane damage can improve intestinal inflammatory damage in crabs, thereby improving the digestive and immune functions of crab intestines, and ultimately improving the growth performance of juvenile crabs. In addition, the Shewanella putrefaciens Z9 also has antibacterial properties, and can improve the disease resistance of crabs by protecting the body's immune system.

The technical features in the above embodiments 1-3 can be combined arbitrarily, and the combined technical solutions all belong to the protection scope of the present application. The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description are only the principles of the present invention. Variations and improvements, which fall within the scope of the claimed invention. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims (6)

1. The Shewanella putrefaciens is Shewanella putrefaciens Z9, is preserved in China center for type culture Collection, and has the preservation number: CCTCC M2020210, and the preservation date is 6 months and 16 days in 2020.

2. Use of Shewanella putrefaciens according to claim 1 for preparing a bacteriostatic preparation, wherein the bacteriostatic object of the bacteriostatic preparation is one or more of Vibrio anguillarum, aeromonas hydrophila and Staphylococcus aureus.

3. Use of Shewanella putrefaciens according to claim 1 for promoting the growth of crabs.

4. A crab farming feed comprising the Shewanella putrefaciens according to claim 1.

5. The crab feed according to claim 4, wherein the Shewanella putrefaciens is present in the crab feed at a concentration of 10 ⁷ -10 ⁹ CFU/g。

6. A crab culture method, characterized in that the crab culture feed of any one of claims 4 to 5 is used for feeding crabs for 5 to 10 weeks, wherein the feeding is performed 1 to 3 times a day, and the feeding is performed according to 4 percent of the body weight each time.

Images