CN111394275A - Bacillus amyloliquefaciens and application thereof, aquatic feed and aquaculture method - Google Patents
Bacillus amyloliquefaciens and application thereof, aquatic feed and aquaculture method Download PDFInfo
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- CN111394275A CN111394275A CN202010109464.1A CN202010109464A CN111394275A CN 111394275 A CN111394275 A CN 111394275A CN 202010109464 A CN202010109464 A CN 202010109464A CN 111394275 A CN111394275 A CN 111394275A
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- bacillus amyloliquefaciens
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- feed
- fishes
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Abstract
The invention discloses a Bacillus amyloliquefaciens strain, which is characterized in that the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens (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, 2 and 18. 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
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 bacillus amyloliquefaciens strain, wherein the bacillus amyloliquefaciens strain is bacillus amyloliquefaciens (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 the fat deposition of fish.
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 concentration of the bacillus amyloliquefaciens SS1 in the aquatic feed 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, 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.
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 shows the growth of Bacillus amyloliquefaciens SS1 according to the present invention on starch medium, the hemolysis, the Aeromonas hydrophila-inhibiting, and the growth on L B 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 shows the effect of Bacillus amyloliquefaciens SS1 of the present invention on fish abdominal fat factor (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 was positive, as shown in FIG. 1, the colonies after 12h of inverted culture on L B medium plates at 28 ℃ were irregular white circles with dry surfaces and volcano-like white bumps in the center of the colonies, similar to a fried egg.
3. Enzyme producing Properties
1) A single colony of SS1 was dipped with a toothpick and spotted on a starch culture medium, after 12 hours of inverted culture at 28 ℃, Lugol iodine solution was dropped on the culture plate, and the plate was left to stand for 3 to 5 minutes, and it was observed that SS1 was able to produce a transparent circle on the starch culture medium, and the result is shown in FIG. 2 ①, thus demonstrating that SS1 can produce amylase in vitro.
2) Method for measuring amylase activity by DNS reducing sugar method
A standard glucose solution (2mg/m L) is prepared by accurately weighing 0.2g glucose, dissolving in distilled water, accurately metering to 100m L with volumetric flask, mixing, and storing in refrigerator at 4 deg.C.
3, 5-dinitrosalicylic acid (DNS) reagent preparation 6.3g of DNS and 262m L2 mol/L NaOH solution are added to a 500m L hot aqueous solution containing 185g of sodium potassium tartrate, 5g of crystalline phenol and 5g of Na are added2SO3Stirring to dissolve, cooling, adding distilled water to a constant volume of 1L, and storing in a brown bottle for one week in dark place.
The specific operation method comprises the following steps:
6 clean graduated test tubes are respectively numbered, glucose solutions with the concentrations of 0mg/m L, 0.4mg/m L0, 0.8mg/m L, 1.2mg/m L, 1.6mg/m L and 1mg/m L are respectively prepared by using a standard glucose solution (2mg/m L), the glucose solutions with different concentrations of 1m L are respectively mixed with 1.5m L DNS reagent, the mixture is immediately placed on ice after being heated in boiling water bath for 5min, finally distilled water is used for fixing the volume to 10m L, the absorbance value of the reaction mixture is measured at OD 540nm after the mixture is vertically reversed and mixed, and finally, the absorbance value (540 OD) is taken as an abscissa and the glucose concentration (mg/m L) is taken as an ordinate for drawing to draw a glucose standard curve.
Taking 1m L crude enzyme solution (namely SS1 bacterial solution) and 1m L culture medium (used as a contrast) to be respectively and uniformly mixed with a substrate solution (1% soluble starch solution) and then react in a water bath at 45 ℃ for 30min, establishing three parallels, immediately absorbing 1m L reaction solution and mixing with 1.5m L DNS reagent after the reaction is finished, immediately placing on ice after boiling water bath is 5min, finally, using distilled water to fix the volume to 10m L, and measuring the absorbance value of the reaction mixture at OD 540nm after the mixture is vertically inverted and uniformly mixed.
The glucose standard curve is: y is 0.1227x-0.0073 (R)2＝0.9938)
Amylase activity (U/m L) ═ 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
The standard is prepared by diluting acetic acid, propionic acid, and butyric acid with distilled water to 1000 times of mixed standard acid, adding 100 μ L50% sulfuric acid, acidifying, vortexing for 30s, adding 400 μ L diethyl ether, vortexing for 15s, standing for 2min, extracting, centrifuging at 4 deg.C and 12000rpm/min for 5min, and collecting the upper organic phase into 1m L brown vial.
Sample preparation, using 200 mu L SS1 bacterial liquid or corresponding culture medium as a control, adding 100 mu L50% sulfuric acid for acidification, carrying out vortex oscillation for 30s, adding 400 mu L diethyl ether, carrying out vortex oscillation for 15s, standing for 2min for extraction, centrifuging for 5min at 4 ℃ and 12000rpm/min, and absorbing an upper organic phase into a 1m L brown vial.
The gas chromatography conditions comprise initial temperature of the chromatographic column of 100 deg.C, holding for 2min, 5 deg.C raising to 180 deg.C per minute, holding for 2min, controlling flow rate of the chromatographic column to be 1m L/min, injection port temperature of 220 deg.C, no shunt, injection of 1 μ L, detector temperature of 200 deg.C, air of 200m L/min, hydrogen of 32m L/min, and nitrogen of 24m L/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 L B liquid culture medium according to the ratio of 1:1000 respectively for resuscitation, shaking the strain for 12h under the conditions of 28 ℃ and 220rpm/min, dipping the Aeromonas hydrophila strain liquid by using an inoculating loop, streaking on a L B culture medium plate, carrying out inverted culture at 28 ℃ for 12h, dipping an SS1 single colony by using a toothpick, spotting on a blood plate, and carrying out inverted culture at 28 ℃ for 12 h.
As shown in fig. 2 ②, SS1 (i.e., "1" in fig. 2 ②) did not cause hemolysis on the blood plate, while pathogenic bacteria a.hydcs4 (i.e., "2" and "3" in fig. 2 ②) produced a hemolysis loop on the blood plate, thereby indicating that SS1 is not significantly pathogenic and has high safety.
6. In vitro bacteriostatic properties
The SS1 bacterial liquid was adjusted to a concentration of 1 × 10 with PBS buffer6CFU/m L, centrifuging the Aeromonas hydrophila solution at 12000rpm/min, collecting precipitate, resuspending with PBS buffer solution, and adjusting concentration to 1 × 109CFU/m L, spreading 100 μ L on L B culture medium, drying the surface of the culture medium, perforating the culture medium with a perforator, picking out excess agar with a sterile needle, adding SS1 bacterial solution 20 μ L into the holes, and culturing in 28 deg.C incubator for 12 h.
From FIG. 2 ③, it can be observed that SS1 can inhibit the growth of Aeromonas hydrophila, and produce a bacteriostatic transparent zone, which indicates that the bacterium has a certain bacteriostatic action.
7. Stress resistance characteristic
Preparing L B solid culture medium containing 0.3% of ox bile salt, dipping a single colony of SS1 on L B solid culture medium with a sterile toothpick, spotting on L B solid culture medium containing 0.3% of ox bile salt, and performing inverted culture at 28 ℃ for 12 h.
From FIG. 2 ④, it can be observed that SS1 can grow on L B solid medium containing 0.3% of bovine bile salt, and produce transparent circles resistant to bile salt, indicating that the bacterium has better bile salt resistance.
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, tryptone 5 g/L, Yeast extract 2 g/L2PO42g/L，MgSO4·7H2Preparing a starch culture medium by using O2 g/L5 g/L, soluble starch 10 g/L and agarose 20 g/L, and pouring the mixture in a super clean bench after autoclaving to obtain a starch culture medium solid plate;
s2, mixing intestinal contents of fish (such as tilapia) with PBS buffer solution uniformly according to a ratio of 1: 9(w/v, g/ml), centrifuging at 1500rpm/min, and coating supernatant fluid of 100 mu L on 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, preserving and numbering the inclined planes of the screened colonies, 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;
s2 and SS1 bacterial liquid preparation, namely inoculating the bacillus amyloliquefaciens SS1 into L B liquid culture medium according to the ratio of 1:1000(w/v, g/ml) for resuscitation, shaking the bacteria for 12 hours at 28 ℃ and 220rpm/min to obtain resuscitative bacterial liquid, inoculating the resuscitative bacterial liquid into L B liquid culture medium according to the ratio of 1:100(v/v, ml/ml) for amplification culture, shaking the bacteria for 24 hours at 28 ℃ and 220rpm/min, centrifuging for 20 minutes at 4 ℃ and 12000rpm/min, collecting thalli, and carrying out heavy suspension by PBS buffer solution to obtain heavy-suspension bacterial liquid;
s3, preparing feed: uniformly mixing the heavy-suspension bacterium liquid and high-starch feed (containing 45% of starch in a weight ratio) in a ratio of 1:2(v/w, ml/g) to ensure that the concentrations of the bacillus amyloliquefaciens SS1 in the feed are respectively 105CFU/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 ratio (%) (end weight-initial weight) is × 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 data, namely 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
Each group of 5 fishes was sampled to prepare a glucose solution of 500mg/m L, glucose concentration was injected intraperitoneally at 200mg/kg per tail, blood was collected at 0h, 0.5h, 1.5h and 3h, serum was collected, blood glucose concentration was measured with a glucose kit and 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, x: 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 data, namely 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
Measuring the fat content of the liver by a chloroform-methanol method, namely measuring the fat content of the liver by 3 treatment groups, wherein each group comprises 6 parallel groups, two groups of 36 clean glass test tubes with scales of 10ml are needed, the groups are marked and weighed and peeled, about 0.5g of liver sample is added into each tube, 6ml of chloroform-methanol (chloroform: methanol is 2: 1) is added into a ventilation kitchen, a tube cover is screwed down, the tubes are kept stand overnight at 4 ℃, the sample is taken out and vibrated for 30s on a vortex oscillator, the tubes are placed into a refrigerator at 4 ℃ for standing for 1h, then 0.37 mol/L mol potassium chloride is added into the ventilation kitchen, the tubes are centrifuged at 1500rpm/min for 10min in a low-speed centrifuge after being vibrated again, a bottom layer transparent liquid is sucked out by a pasteur suction tube, the tubes are transferred into a new weighed and marked glass tube after being filtered by a funnel, filter paper is washed by trichloromethane, the test tubes are placed into a vacuum drying oven, after the trichloromethane is volatilized, the glass tubes are taken out, the tube weight is weighed, the tube weight is obtained by subtracting the tube weight recorded before the fat content of the.
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 significantly increased compared to the CON group, but the liver fat content and the liver TG content of the HCB group were significantly decreased compared to the HCD group, and further, the HE section of the liver of fish and the evaluation of liver fat area (L ipid simple area of liver) showed that the size of the fat cells and the liver fat area of the HCB group were significantly decreased compared to 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 were removed, weighed and recorded, the capsid ratio and capsid protein content were calculated, the protein content was determined by kjeldahl method and the expression level of protein synthesis marker gene mTOR was detected by real-time fluorescence quantification method, the results are shown in fig. 9.
The results show that the body-shell ratio of the CON group and the HCD group is not obviously different, but the body-shell ratio of the HCB group is obviously increased, which indicates that the protein content is obviously 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 data, namely HCB1, HCB2 and HCB 3.
Therefore, the addition of the bacillus amyloliquefaciens SS1 can activate protein synthesis pathways by increasing the expression level 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 L B liquid culture medium for resuscitation, shaking at 28 deg.C and 220rpm/min for 12h, inoculating resuscitation bacteria liquid into L B 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/m L, taking 21 fishes (such as tilapia) per group, and performing challenge experiment, wherein each fish is injected into abdominal cavity with 10 injections6CFU/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 body head kidney macrophage, separating head 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/m L measurement of oxygen respiratory burst activity of head and kidney macrophages absorbance was measured at 540nm using NBT method, 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 (10)
1. The Bacillus amyloliquefaciens is characterized by being Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SS1 and being deposited in China center for type culture collection with the deposit numbers as follows: CCTCC M2020024, and the preservation date is 2020, 2 and 18.
2. Use of the bacillus amyloliquefaciens SS1 of claim 1 in the preparation of a bacteriostatic formulation.
3. Use of the bacillus amyloliquefaciens SS1 according to claim 1 for promoting fish growth.
4. Use of the bacillus amyloliquefaciens SS1 according to claim 1 for improving blood glucose homeostasis in fish.
5. Use of the bacillus amyloliquefaciens SS1 according to claim 1 for reducing fat deposits in fish.
6. Use of the bacillus amyloliquefaciens SS1 according to claim 1 for promoting the deposition of fish body proteins.
7. The use of the bacillus amyloliquefaciens SS1 as claimed in claim 1 for improving the disease resistance of fish.
8. An aquaculture feed comprising bacillus amyloliquefaciens SS1 of claim 1.
9. The aquaculture feed product of claim 10 wherein said bacillus amyloliquefaciens SS1 is present in said aquaculture feed product at a concentration of 105-107CFU/g。
10. A method of aquaculture wherein fish are fed with an aquaculture feed as claimed in claims 8 to 9 1 to 2 times per day at a rate of 4% by weight for a period of 5 to 10 weeks.
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