CN111394275B

CN111394275B - Bacillus amyloliquefaciens and application thereof, aquatic feed and aquaculture method

Info

Publication number: CN111394275B
Application number: CN202010109464.1A
Authority: CN
Inventors: 张美玲; 许蓉; 杜震宇; 黄清
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2020-02-22
Filing date: 2020-02-22
Publication date: 2022-05-27
Anticipated expiration: 2040-02-22
Also published as: CN111394275A

Abstract

The invention discloses a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), which is characterized in that the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens SS1 and is preserved in China center for type culture collection with the preservation number as follows: CCTCCM2020024 with preservation date of 2020, 1 month and 7 days. The bacillus amyloliquefaciens is high in safety, and has the functions of improving the utilization rate of carbohydrates of fishes, promoting the growth of the fishes, improving the blood sugar steady state of the fishes, relieving abnormal deposition of fat of the fishes, increasing the deposition of body protein, improving the disease resistance and the like.

Description

Bacillus amyloliquefaciens and application thereof, aquatic feed and aquaculture method

Technical Field

The invention relates to the field of aquaculture, in particular to bacillus amyloliquefaciens and application thereof, an aquaculture feed and an aquaculture method.

Background

With the continuous expansion of aquaculture scale and the annual increase of yield in China, fish meal and fish oil resources are increasingly in short supply, so that the improvement of the utilization efficiency of the existing feed raw materials is an important direction for relieving the shortage of the feed raw materials. Carbohydrates are one of the important metabolic energy supply substrates for fish and are also a cheaper energy source in aquatic feeds. Researches show that the addition of a proper amount of starch in the feed is not only beneficial to the forming of feed particles, but also beneficial to saving feed protein, reducing feed cost and reducing nitrogen emission of fish bodies. However, compared with higher animals, the digestion and utilization efficiency of the fish to the carbohydrate such as starch is lower, and the nutritional diseases such as the growth inhibition, the low feed utilization efficiency, the abnormal fat deposition, even the high death rate and the like of the fish body can be caused by taking the feed with high carbohydrate level for a long time. Therefore, the utilization efficiency of the carbohydrates by the fishes is improved, the growth of the fishes can be promoted, the feed protein is saved, the culture cost is reduced, and the economic benefit is improved.

In recent years, many studies have shown that fish gut microorganisms play an important role in promoting host health. The intestinal microorganisms not only affect the digestion of feed, the absorption of nutrient substances and the energy supply, but also regulate and control the normal physiological functions of a host and the occurrence and development of diseases. Therefore, as a substitute for antibiotics, probiotics are widely applied to the fields of livestock breeding, aquaculture, medicine and the like, can inhibit the growth of harmful bacteria, promote the development of immune system, produce beneficial metabolites, provide nutrients and energy for organisms, regulate metabolic disorders, protect intestinal mucosa barriers and the like.

At present, in the aquaculture process, probiotics are added into feed or water, so that researches for adjusting the metabolic stability of organisms and improving disease resistance are increasing, wherein bacillus and lactic acid bacteria are used more frequently. The bacillus is one of the most abundant and important strains of active products in a microorganism bank, has short growth period and very high propagation speed, and produces various metabolites in the growth and propagation processes.

However, the efficacy of the bacillus for aquatic products is mainly focused on improving disease resistance, and the effect of the bacillus for aquatic products on the regulation of fish metabolism, particularly on the aspect of promoting the utilization of carbohydrates in fish bodies, is rarely reported.

Disclosure of Invention

The bacillus amyloliquefaciens is high in safety and has the functions of improving the utilization rate of carbohydrates of fishes, promoting the growth of the fishes, improving the blood sugar steady state of the fishes, relieving abnormal deposition of fats of the fishes, increasing the deposition of body proteins, improving the disease resistance and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

provides a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), wherein the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens SS1 and is preserved in China center for type culture collection with the preservation number as follows: CCTCC M2020024, and the preservation date is 1 month and 7 days in 2020.

Provides an application of the bacillus amyloliquefaciens SS1 in preparing a bacteriostatic preparation.

Provides an application of the bacillus amyloliquefaciens SS1 in promoting fish growth.

Provides an application of the bacillus amyloliquefaciens SS1 in improving the blood sugar homeostasis of fishes.

Provides an application of the bacillus amyloliquefaciens SS1 in reducing fish fat deposition.

Provides an application of the bacillus amyloliquefaciens SS1 in promoting the protein deposition of fish bodies.

Provides an application of the bacillus amyloliquefaciens SS1 in improving the disease resistance of fish.

An aquatic feed product is provided, which comprises the bacillus amyloliquefaciens SS 1.

Preferably, the feed product is, in said aquatic feed product,the concentration of the bacillus amyloliquefaciens SS1 is 10⁵-10⁷CFU/g。

Provides an aquaculture method, which adopts the aquatic feed to feed fishes 1-2 times per day, wherein the aquaculture time is 5-10 weeks.

The invention has at least the following beneficial effects:

the bacillus amyloliquefaciens SS1 can be added into fish culture feed to improve the utilization efficiency of carbohydrates by fish and improve the digestive enzyme activity to promote growth by the bacillus amyloliquefaciens SS 1; further, said Bacillus amyloliquefaciens SS1 can increase glycolysis level in fish liver, thereby improving blood sugar homeostasis, alleviating abnormal fish fat deposition by increasing energy consumption, and increasing body protein deposition by activating protein synthesis pathway; in addition, the bacillus amyloliquefaciens SS1 also has bacteriostatic property and can improve the disease resistance of fish bodies by protecting the immune system of organisms.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 shows the colony morphology of Bacillus amyloliquefaciens SS1 according to the present invention;

FIG. 2 is a graph showing the growth of Bacillus amyloliquefaciens SS1 of the present invention on a starch medium, the hemolysis, the Aeromonas hydrophila-inhibiting, and the growth on LB solid medium containing 0.3% bovine bile salt;

FIG. 3 is data of the amylase activity produced by Bacillus amyloliquefaciens SS1 of the present invention;

FIG. 4a shows the results of gas chromatographic separation of acetic acid, propionic acid and butyric acid;

FIG. 4b shows the result of gas chromatographic separation of acetic acid produced in vitro by Bacillus amyloliquefaciens SS1 of the present invention;

FIG. 5 shows the effect of Bacillus amyloliquefaciens SS1 of the present invention on the average fish body weight (a), weight gain (b), and feed efficiency (c);

FIG. 6 is a graph showing the effect of Bacillus amyloliquefaciens SS1 of the present invention on fasting blood glucose levels (a), sugar tolerance (b), and AUC (c) in fish;

FIG. 7 is a graph showing the effect of Bacillus amyloliquefaciens SS1 of the present invention on fish abdominal fat index (MFI) (a), serum Triglyceride (TG) (b), abdominal fat cell size (c) and abdominal fat cell size assessment (d);

FIG. 8 is a graph showing the effect of Bacillus amyloliquefaciens SS1 of the present invention on fish liver fat content (a), liver TG (b), liver adipocyte size (c), and liver fat area assessment (d);

FIG. 9 shows the effect of Bacillus amyloliquefaciens SS1 on fish body hull ratio (a), hull protein (b), and mTOR gene expression level (c);

FIG. 10 is a graph showing the effect of Bacillus amyloliquefaciens SS1 of the present invention on fish survival rate (a) and head kidney macrophage oxygen respiratory burst activity (b).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1:

the embodiment provides a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS1 (hereinafter referred to as 'SS 1'), which is preserved in China center for type culture Collection and has the preservation number: CCTCC M2020024, the preservation date is: year 2020, 1, 7.

The information of the above strains is as follows:

1. the source of the strain

The strain is isolated from intestinal tract of healthy tilapia (Oreochromys mossambicaus).

2. Morphological characteristics

Gram staining is positive, as shown in figure 1, colonies which are inversely cultured on an LB medium plate for 12h at 28 ℃ are white irregular circles, the surfaces of the colonies are dry, and volcano-like white bulges are arranged in the centers of the colonies and are similar to omelettes.

3. Enzyme producing Properties

1) Dipping a single colony of SS1 with a toothpick, spotting the colony on a starch culture medium, carrying out inverted culture at 28 ℃ for 12h, then dropwise adding Lugol iodine solution on a culture plate, standing for 3-5min, and observing that SS1 can generate a transparent ring on the starch culture medium, wherein the result is shown in figure 2 (r). This demonstrates that SS1 can produce amylase in vitro.

2) Method for measuring amylase activity by DNS reducing sugar method

Standard glucose solution (2mg/mL) preparation: accurately weighing 0.2g of glucose, dissolving in distilled water, accurately metering to 100mL by using a volumetric flask, uniformly mixing, and storing in a refrigerator at 4 ℃ for later use.

Preparation of 3, 5-dinitrosalicylic acid (DNS) reagent: 6.3g of DNS and 262mL of a 2mol/L NaOH solution were added to 500mL of a hot aqueous solution containing 185g of potassium sodium tartrate, and 5g of crystalline phenol and 5g of Na were added₂SO₃Stirring for dissolving, cooling, adding distilled water to constant volume of 1L, and storing in brown bottle in dark for one week.

The specific operation method comprises the following steps:

taking 6 clean graduated test tubes to be numbered respectively, preparing glucose solutions with the concentrations of 0mg/mL, 0.4mg/mL, 0.8mg/mL, 1.2mg/mL, 1.6mg/mL and 1mg/mL respectively by using a standard glucose solution (2mg/mL), mixing 1mL of glucose solutions with different concentrations with 1.5mL of DNS reagent respectively, heating in a boiling water bath for 5min, immediately placing on ice, finally fixing the volume to 10mL by using distilled water, determining the absorbance value of a reaction mixture at OD 540nm after vertically reversing and uniformly mixing, and finally drawing a glucose standard curve by taking the absorbance value (OD 540) as an abscissa and the glucose concentration (mg/mL) as an ordinate.

Taking 1mL of crude enzyme solution (namely SS1 bacterial solution) and 1mL of culture medium (used as a contrast) to be respectively and uniformly mixed with a substrate solution (1% soluble starch solution) and then reacting for 30min in a water bath at 45 ℃, establishing three parallels, immediately absorbing 1mL of reaction solution and 1.5mL of DNS reagent after the reaction is finished, immediately placing on ice after the boiling water bath is 5min, finally diluting to 10mL with distilled water, and determining the absorbance value of the reaction mixture at OD 540nm after the mixture is inverted and uniformly mixed.

The glucose standard curve is: y is 0.1227x-0.0073 (R)²＝0.9938)

Amylase activity (U/mL) ═ k × OD × 1000 × n/(180 × 30)

Wherein k is the slope of the glucose standard curve; OD is absorbance at 540 nm; 1000 is a unit conversion multiple; n is the dilution multiple; 180 is the molecular weight of glucose; and 30 is reaction time (min).

As shown in FIG. 3, the amylase produced in vitro from SS1 had a high amylase activity (approximately 1.0U/L).

4. Characteristic of acetic acid production

Preparing a standard substance: diluting acetic acid, propionic acid and butyric acid with distilled water to 1000 times of mixed standard acid, adding 100 μ L50% sulfuric acid for acidification, performing vortex oscillation for 30s, adding 400 μ L diethyl ether, performing vortex oscillation for 15s, standing for 2min for extraction, and centrifuging at 4 deg.C 12000rpm/min for 5 min; the upper organic phase was pipetted into a 1mL brown vial.

Sample preparation: taking 200 μ L of SS1 bacterial liquid or corresponding culture medium as control, adding 100 μ L of 50% sulfuric acid for acidification, performing vortex oscillation for 30s, adding 400 μ L of diethyl ether, performing vortex oscillation for 15s, standing for 2min for extraction, and centrifuging at 4 deg.C and 12000rpm/min for 5 min; the upper organic phase was pipetted into a 1mL brown vial.

Gas chromatography conditions: the initial temperature of the chromatographic column is 100 ℃, the temperature is kept for 2min, the temperature is raised to 180 ℃ per minute at 5 ℃, the temperature is kept for 2min, and the flow rate of the chromatographic column is controlled to be 1 mL/min; the temperature of a sample inlet is 220 ℃, no shunt is carried out, and the sample injection amount is 1 mu L; the detector temperature was 200 deg.C, air 200mL/min, hydrogen 32mL/min, nitrogen 24 mL/min.

As shown in FIG. 4a, 3 short-chain fatty acids can be effectively separated by gas chromatography column, wherein the acetic acid retention time is 7.019min, the propionic acid retention time is 8.622min, and the butyric acid retention time is 10.422 min; in addition, as shown in fig. 4b, SS1 shows a relatively distinct peak at 7.020min, which indicates that SS1 can ferment starch in vitro to produce acetic acid, and the acetic acid can provide energy for the body and has an important role in reducing blood sugar level and fat deposition.

5. Pathogenicity

Inoculating the frozen strain SS1 and the separated Aeromonas hydrophila CS4(A.hyd CS4) into an LB liquid culture medium according to a ratio of 1:1000 respectively for resuscitation, shaking the strain at the condition of 28 ℃ and 220rpm/min for 12h, dipping the Aeromonas hydrophila strain liquid by using an inoculating loop, streaking on a LB culture medium plate, carrying out inverted culture at the temperature of 28 ℃ for 12h, dipping an SS1 single colony by using a toothpick, spotting on a blood plate, and carrying out inverted culture at the temperature of 28 ℃ for 12 h.

As shown in FIG. 2, SS1 (i.e. "1" in FIG. 2) has no hemolysis on the blood plate, while pathogenic bacteria A.hyd CS4 (i.e. "2" and "3" in FIG. 2) have hemolysis ring on the blood plate, thus, SS1 has no obvious pathogenicity and high safety.

6. In vitro bacteriostatic properties

The SS1 bacterial solution was adjusted to a concentration of 1X 10 with PBS buffer⁶CFU/mL, centrifuging the Aeromonas hydrophila solution at 12000rpm/min, collecting precipitate, resuspending with PBS buffer solution, and adjusting concentration to 1 × 10⁹CFU/mL, 100. mu.L of the suspension was spread on LB medium, after the surface of the medium was dried, a hole was punched in the medium using a punch, excess agar was picked out using a sterile needle, 20. mu.L of SS1 bacterial solution was added to the hole, and the mixture was cultured in a 28 ℃ incubator for 12 hours.

From figure 2, it can be observed that SS1 can inhibit the growth of aeromonas hydrophila, and generate a bacteriostatic transparent circle, which shows that the bacterium has a certain bacteriostatic action.

7. Stress resistance characteristic

Preparing LB solid culture medium containing 0.3% of ox bile salt, dipping a single colony of SS1 on the LB solid culture medium by using a sterile toothpick, spotting the single colony on the LB solid culture medium containing 0.3% of ox bile salt, and carrying out inverted culture at 28 ℃ for 12 h.

From FIG. 2, it can be observed that SS1 can grow in LB solid medium containing 0.3% of bovine bile salt, producing a bile salt resistant transparent ring, which indicates that the bacterium has better bile salt resistant ability.

Example 2:

this example provides a method of isolation of Bacillus amyloliquefaciens SS1 (hereinafter "SS 1") described in example 1, comprising the steps of:

s1, 5g/L tryptone, 2g/L yeast extract, KH₂PO₄2 g/L，MgSO₄·7H₂Preparing a starch culture medium by using a mixture ratio of O2 g/L, NaCl 5g/L, soluble starch 10g/L and agarose 20g/L, and inverting the plate in a super clean bench after autoclaving to obtain a starch culture medium solid plate;

s2, taking intestinal contents of fishes (such as tilapia) and PBS buffer solution according to the weight ratio of 1: 9(w/v, g/ml), centrifuging at 1500rpm/min, and spreading 100 μ L of supernatant onto the starch culture medium solid plate;

s3, carrying out inverted culture at 28 ℃ for 24h, then screening colonies according to the size, shape and color of the colonies, scoring inclined planes on the screened colonies, storing and numbering the inclined planes, and carrying out 16S rRNA molecular identification on the screened strains to obtain the bacillus amyloliquefaciens SS 1.

Example 3:

this example provides a method for aquaculture using bacillus amyloliquefaciens SS1 (hereinafter referred to as "SS 1") described in example 1, which specifically includes the following steps:

s1, temporarily culturing the fry: purchasing 600 tails of fish fries (such as tilapia fries), temporarily culturing for 2 weeks by using commercial feed, then putting into aerated water at 28 ℃ for temporary culture, changing water every two days, and ending the temporary culture after the fish fries adapt to a growth environment and no diseases are determined; the fry is divided into five groups, namely a normal group (CON), a high starch group (HCD), a probiotic group 1(HCB1), a probiotic group 2(HCB3) and a probiotic group 3(HCB3), wherein each group comprises three parallel groups, each group comprises 30 parallel tails, and the weight of each group is 1.6 +/-0.1 g;

preparing S2 and SS1 bacterial liquid: inoculating the bacillus amyloliquefaciens SS1 into an LB liquid culture medium according to a ratio of 1:1000(w/v, g/ml) for recovery, and shaking the bacillus amyloliquefaciens for 12 hours at 28 ℃ and 220rpm/min to obtain a recovery bacterium solution; inoculating resuscitation bacteria liquid into LB liquid culture medium according to a ratio of 1:100(v/v, ml/ml) for amplification culture, shaking bacteria for 24h at 28 ℃ and 220rpm/min, centrifuging for 20min at 4 ℃ and 12000rpm/min, and collecting thalli; resuspending the suspension with PBS buffer solution to obtain a resuspended bacterial liquid;

s3, preparing feed: mixing the re-suspended bacteria liquid with the high-concentration liquidThe starch feed (45% starch by weight) is mixed uniformly according to the ratio of 1:2(v/w, ml/g) to make the concentration of Bacillus amyloliquefaciens SS1 in the feed be 10 respectively⁵CFU/g、10⁶CFU/g、10⁷CFU/g to obtain probiotic feed (namely aquatic feed) with three different strain concentrations, and then granulating the probiotic feed;

s4, feeding: the normal group (CON), the high starch group (HCD) and the probiotic group 1(HCB1) (feeding concentration 10) were fed with commercial feed, high starch feed (containing 45% starch by weight) and three probiotic feeds with different strain concentrations⁵CFU/g probiotic feed), probiotic group 2(HCB3) (fed at a concentration of 10⁶CFU/g probiotic feed), probiotic group 3(HCB3) (fed at a concentration of 10⁷CFU/g probiotic feed) fry, fed 1-2 times per day, and fed 4% of the body weight each time; starving for 24h after 5-10 weeks (preferably 8 weeks) of culture, and weighing, collecting blood and collecting tissue samples of 10 fish in each group.

The commercial feed, the high starch feed (containing 45% by weight of starch) and the probiotic feed raw material formula were tested and shown in table 1.

TABLE 1 feed ingredient formulation Table (g)

Wherein: 1. complex (mg or IU/kg): vitamin A: 500,000i.u. (international units); vitamin D3: 50,000 i.u.; vitamin E: 2500 mg; vitamin K3: 1000 mg; vitamin B1: 5000 mg; vitamin B2: 5000 mg; vitamin B6: 5000 mg; 125000 μ g of vitamin B; inositol: 25,000 mg; folic acid: 1000 mg; pantothenic acid: 10,000 mg; biotin: 250 mg; choline: 100,000 mg; nicotinic acid: 25,000 mg; vitamin C: 10,000 mg.

2. Remineralization (g/kg): calcium carbonate: 314.0 g; potassium dihydrogen phosphate: 469.3 g; magnesium sulfate heptahydrate: 147.4 g; sodium chloride: 49.8 g; ferrous gluconate: 10.9 g; 3.12g of manganese sulfate monohydrate; ammonium molybdate: 0.06 g; zinc sulfate heptahydrate: 4.67 g; anhydrous copper sulfate: 0.62 g; cobalt chloride hexahydrate: 0.08 g; sodium selenite: 30.02 g.

The function of the bacillus amyloliquefaciens SS1 is as follows:

(1) SS1 function of promoting fish growth (including weight gain)

After the culture is finished, the total weight of each group of fish bodies is respectively weighed, and the average weight, the weight gain rate and the bait coefficient of each group are calculated.

Wherein, the average weight (g/tail) is total weight (g)/mantissa, and the weight gain (%) is (end weight-initial weight) × 100/initial weight.

As shown in fig. 5(a), compared with the CON group, the average weight of the HCD group was significantly increased, and the average weight of the HCB group was also significantly increased, compared with the HCD group, and as shown in fig. 5(b), the weight gain rate of the CON group was not significantly different from that of the HCD group, but the weight gain rate of the HCB group was significantly increased, compared with that of the HCD group, which indicates that the bacillus amyloliquefaciens SS1 of the present invention can significantly and rapidly promote the weight gain of fish bodies. Further, as shown in FIG. 5(c), the bait ratio of the HCB group was decreased compared to the CON group and the HCD group, and was particularly decreased significantly compared to the HCD group,

as described above, Bacillus amyloliquefaciens SS1 has high amylase activity and can improve the utilization rate of carbohydrates, so that the digestive enzyme activity, such as the activity of amylase, can be improved by applying the Bacillus amyloliquefaciens SS1 in aquatic feeds, the growth of fish bodies can be promoted, the cost can be reduced, and the economic benefit can be increased.

Note: in fig. 5 "+" represents the difference between groups, and: p < 0.05, x: p < 0.01, x: p is less than 0.001, and the HCB group data in (a) - (c) are the average values of three groups of HCB1, HCB2 and HCB 3.

(2) Function of bacillus amyloliquefaciens SS1 on improving blood sugar homeostasis of fish bodies

1) Blood was collected, serum was collected, and fasting blood glucose concentration was measured with a glucose kit.

2) Sugar tolerance test

5 fishes in each group were taken to prepare a 500mg/mL glucose solution, the intraperitoneal injection of each fish was performed at a glucose concentration of 200mg/kg, blood was collected at 0h, 0.5h, 1.5h and 3h, serum was collected, the blood glucose concentration was measured with a glucose kit and the area under the curve (AUC) was calculated, and the results are shown in FIG. 6.

As can be seen from fig. 6(a), fasting blood glucose levels were significantly increased in the HCD group compared to the CON group, but were significantly decreased in the HCB group compared to the HCD group, indicating that blood glucose levels in fish bodies could be significantly decreased by the addition of bacillus amyloliquefaciens SS1, while the results of the sugar tolerance experiment in fig. 6(b) and AUC in fig. 6(c) demonstrate that fish bodies in the HCB group had stronger sugar tolerance.

Therefore, the glycolysis level of the liver of the fish can be increased by using the bacillus amyloliquefaciens SS1, and the blood sugar level in the fish can be reduced by producing acetic acid, so that when the fish ingests the feed with high carbohydrate level for a long time, the feed helps to stabilize the blood sugar level in the fish, improve the sugar tolerance and improve the blood sugar steady state of the fish.

Note: in fig. 6 "+" represents the difference between groups, and: p < 0.05, x: p < 0.01,.: p is less than 0.001; "#" in fig. 6(b) represents the difference between the HCB group and the HCD group, and #: p < 0.05, # #: p < 0.01, ###' #: p is less than 0.001, and the HCB group data in (a) - (c) are the average values of three groups of HCB1, HCB2 and HCB 3.

(3) Bacillus amyloliquefaciens SS1 for reducing fat deposition in liver and abdominal cavity of fish

1) Placing adipose tissues with the same parts and weight of different groups of fishes in a Bonn's stationary liquid for preparing HE slices, collecting abdominal adipose tissues and weighing; liver tissues of the same part and weight were placed in Bonn's fixative for HE slice preparation, and liver tissues were collected and weighed.

2) HE slice

A conventional paraffin section preparation method and HE dyeing.

3) Liver fat content assay and TG assay

Determining the fat content of the liver by a chloroform-methanol method: for 3 treatment groups, 6 in each group, two sets of 36 10ml clean glass tubes with a scale were used, and after marking, the tubes were weighed and peeled off, about 0.5g of liver sample was added to each tube, 6ml of chloroform-methanol (chloroform: methanol 2: 1) was added to the tube in a fume hood, the tube was closed and left to stand overnight at 4 ℃. Taking out a sample, oscillating the sample on a vortex oscillator for 30s, placing the sample in a refrigerator with the temperature of 4 ℃ for standing for 1h, then adding 0.37mol/L potassium chloride into a ventilation kitchen, after oscillation again, centrifuging the sample in a low-speed centrifuge at 1500rpm/min for 10min, sucking out the lower layer transparent liquid by using a Pasteur suction pipe, transferring the lower layer transparent liquid into a new weighed and marked glass tube after filtering the lower layer transparent liquid by a funnel, flushing filter paper by using trichloromethane, placing the test tube into a vacuum drying box, taking out the glass tube after the trichloromethane is volatilized, placing the glass tube into a dryer, cooling the glass tube, weighing the tube weight, subtracting the tube weight recorded before to obtain the fat weight, and calculating the fat content of the liver.

Serum and liver TG detection was performed according to Nanjing kit instructions.

As can be seen from fig. 7, the CON group did not have a significant difference in celiac adipose index (MFI) from the HCD group, but the HCB group had a significant decrease in MFI compared to the HCD group; and the serum TG in the HCD group is obviously increased compared with the CON group, but the serum TG in the HCB group is obviously reduced compared with the HCD group; the results of the HE sections of abdominal adipose tissues of fish and the evaluation of the size of abdominal adipocytes (Average area value of adipocyte) demonstrated that the size of abdominal adipocytes was significantly increased in the HCD group compared to the CON group, but significantly decreased in the HCB group compared to the HCD group.

Further, as can be seen from fig. 8, the liver fat content and the liver TG content of the HCD group were both significantly increased compared to the CON group, but the liver fat content and the liver TG content of the HCB group were both significantly decreased compared to the HCD group; in addition, the results of the HE section of the liver of a fish body and the evaluation of the fat area of the liver (Lipid drop area of liver) show that the size of fat cells and the fat area of the liver are remarkably reduced in the HCB group compared with the HCD group.

Note: in fig. 7-8 "", the differences between groups are represented, and: p < 0.05, x: p < 0.01, x: p is less than 0.001, and the HCB group data in (a), (b) and (d) is the average value of three groups of HCB1, HCB2 and HCB 3; the HE slice of HCB group in FIG. 7(c) was HCB3 group (SS1 concentration: 10)⁷CFU/g) abdominal adipose tissue sections of fish, HE sections of HCB group in FIG. 8(c) were HCB3 group (SS1 concentration of 10)⁷CFU/g) liver sections of tilapia.

Therefore, the addition of the bacillus amyloliquefaciens SS1 can remarkably reduce the TG content of the liver and abdominal fat of the fish by promoting the utilization capacity of carbohydrates (such as starch) of fish bodies and increasing energy consumption so as to avoid abnormal deposition of the liver and abdominal fat of the fish.

(4) Effect of Bacillus amyloliquefaciens SS1 on fish protein deposition

The head, tail and internal organs of each group of fish bodies are removed, the fish bodies are weighed and recorded, the hull ratio and the hull protein content are calculated, the protein content is determined by adopting a Kjeldahl method, and the expression quantity of the protein synthesis marker gene mTOR is detected by adopting a real-time fluorescence quantitative method, and the result is shown in figure 9.

The result shows that the body-shell ratio of the CON group and the HCD group has no significant difference, but the body-shell ratio of the HCB group is obviously increased, which indicates that the protein content is significantly increased; meanwhile, the results of measuring the capsid protein content and expressing the mTOR show that compared with the HCD group, the capsid protein content and the mTOR expressing amount of the HCB group are obviously increased.

Note: in fig. 9 "+" represents the difference between groups, and: p < 0.05, x: p < 0.01, x: p is less than 0.001, and the HCB group data in (a) - (c) are the average values of three groups of HCB1, HCB2 and HCB 3.

Therefore, the addition of the bacillus amyloliquefaciens SS1 can activate protein synthesis pathways by increasing the expression amount of protein synthesis related genes such as mTOR, further improve the total protein synthesis amount and increase the body protein deposition.

(5) Bacillus amyloliquefaciens SS1 for improving disease resistance of fish

1) Challenge test

Inoculating Aeromonas hydrophila CS4(A.hyd CS4) into LB liquid culture medium for resuscitation, shaking at 28 deg.C and 220rpm/min for 12h, inoculating resuscitated bacterial liquid into LB liquid culture medium at a ratio of 1:100 for amplification culture, shaking at 28 deg.C and 220rpm/min for 24h, centrifuging at 4 deg.C and 12000rpm/min for 20min, collecting thallus, re-suspending with PBS buffer solution, and adjusting concentration to 10⁹CFU/mL. 21 fishes (such as tilapia) are taken from each group for toxicity attack experiment, and 10 percent of enterocoelia is injected into each fish⁶CFU/g, death was observed and recorded for 7 days, and survival was calculated.

2) Breath burst test

Collecting 5 fish per group, anesthetizing with MS-222, separating fish head and kidney macrophage, separating head and kidney macrophage by Percoll density gradient method, detecting and counting cell activity with trypan blue to ensure that the number of living cells is more than 90%, and adjusting cell concentration to 1 × 10⁷cell/mL. The oxygen respiratory burst activity of the macrophages of the head and kidney is measured by an NBT method and an absorbance value is measured at 540 nm. The results are shown in FIG. 10.

The results show that the survival rate of the HCD group is lowest, the survival rate of the HCB group is obviously increased relative to that of the HCD group by adding SS1, and the survival rate can be basically recovered to be the same as that of the CON group after 7 d; the oxygen respiration burst result of the macrophages of the head and kidney shows that the inhalation burst activity of the CON group and the inhalation burst activity of the HCD group have no obvious difference, and compared with the HCD group, the inhalation burst activity of the HCB group is obviously increased.

Note: in fig. 10 "+" represents the difference between groups, and: p < 0.05, x: p < 0.01, x: p is less than 0.001, and the HCB group data in (a) - (b) are the average values of HCB1, HCB2 and HCB 3.

Therefore, the bacillus amyloliquefaciens SS1 can effectively improve the activity of immune cells of fish bodies, promote the infection capacity of the fish against virus and germs (such as aeromonas hydrophila), enhance the comprehensive immunity and disease resistance of the fish bodies and further improve the survival rate.

In conclusion, the bacillus amyloliquefaciens SS1 separated by the method can be used as a safety functional feed additive to be added into high-starch fish feed, so that the utilization efficiency of carbohydrates in fish is improved, and the enzyme activity of digestive enzymes is improved by the bacillus amyloliquefaciens SS1 to promote growth; further, the Bacillus amyloliquefaciens SS1 can increase glycolysis level of liver of fish, thereby improving blood sugar steady state, relieving abnormal deposition of fat of fish by increasing energy consumption, and increasing body protein deposition by activating protein synthesis pathway; in addition, the bacillus amyloliquefaciens SS1 also has bacteriostatic property and can improve the disease resistance of fish bodies by protecting the immune system of organisms.

The technical features of the above embodiments 1 to 3 can be combined arbitrarily, and the combined technical solutions all belong to the protection scope of the present application. The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. Bacillus amyloliquefaciens strainBacillus amyloliquefaciens) The bacillus amyloliquefaciens is bacillus amyloliquefaciens SS1 and is preserved in China center for type culture Collection with the preservation number as follows: CCTCC M2020024, and the preservation date is 2020, 1, 7.

2. Use of the bacillus amyloliquefaciens SS1 of claim 1 in the preparation of a bacteriostatic formulation.

3. Use of the bacillus amyloliquefaciens SS1 of claim 1 for promoting fish growth.

4. Use of the bacillus amyloliquefaciens SS1 of claim 1 for promoting the deposition of fish body protein.

5. An aquaculture feed comprising bacillus amyloliquefaciens SS1 of claim 1.

6. The aquaculture feed product of claim 5 wherein said Bacillus amyloliquefaciens SS1 is present in said aquaculture feed product at a concentration of 10⁵-10⁷CFU/g。