CN109464470B - Microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome as well as preparation method and application thereof - Google Patents
Microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome as well as a preparation method and application thereof. The preparation method of the microbial inoculum for preventing and treating the white feces syndrome of the litopenaeus vannamei comprises the following steps of sequentially carrying out shake flask culture, primary seed culture, secondary seed culture and fermentation culture on enterococcus faecalis, and then drying to prepare the microbial inoculum for preventing and treating the white feces syndrome of the litopenaeus vannamei. The application of the microbial inoculum for preventing and treating the white feces syndrome of the litopenaeus vannamei comprises the steps of mixing the microbial inoculum into feed according to a ratio, and feeding the litopenaeus vannamei in a pond which generates (treats) or does not generate (prevents) the white feces by using the feed. The enterococcus faecalis microbial inoculum can obviously inhibit the growth and the propagation of pathogenic bacteria such as vibrio cholerae, edwardsiella tarda, aeromonas hydrophila and the like, has broad-spectrum antibacterial activity, can prevent and treat white feces of litopenaeus vannamei, and has the prevention and treatment effect of more than 78 percent and obvious effect.
Description
Technical Field
The invention belongs to the technical field of microbial agent disease prevention, and relates to a microbial agent for preventing and treating litopenaeus vannamei white feces syndrome, and a preparation method and application thereof.
Background
The yearbook data of the Chinese aquatic fishery in 2017 shows that the yield of the litopenaeus vannamei in 2016 is 93.23 ten thousand tons, however, the frequent occurrence of white feces syndrome of the litopenaeus vannamei in 2010 is one of the major problems affecting the sustainable development of the prawn aquaculture industry. The main symptoms of litopenaeus vannamei white feces syndrome are: the feces of the sick prawns are thin, white (such as cotton threads), sticky, and abundant and concentrated in the air opening floating under the water surface, and have bad smell. At present, litopenaeus vannamei is mainly treated by antibiotics such as enrofloxacin and the like or combined with some traditional Chinese medicines (such as sanhuangsan) and the like, and all the treatment preparations belong to medicine products, so that the use cost is high, the environmental hazard is large, and the prevention hazard is larger. The difficulty of controlling the disease by farmers is high, the cost of controlling the disease is high, and huge economic loss is caused to the breeding of the litopenaeus vannamei.
There are three possible causes for the formation of this symptom:
(1) the shrimp body is stimulated by certain factors, and is considered to be stimulated by certain toxin of bacteria to trigger the inflammatory reaction of the epithelial cells of the litopenaeus vannamei. Firstly, the glandular epithelium and the intestinal epithelium begin to disintegrate, and hyperplastic cells appear under the basement membrane of the intestinal epithelium. Along with the lapse of time, the number of the hyperplastic cells is increased, the hyperplastic cell layer is thicker and thicker, and the intestinal epithelium is gradually replaced and completely shed; necrotic exfoliated cells, either epithelial or hyperplastic, fall into the intestinal lumen and are excreted in vitro, i.e., "white feces" seen by the naked eye.
(2) The Caochitong and the like think that the pathogenesis of the disease is probably the damage of intestinal wall and mucous membrane caused by the large growth and proliferation of vibrio cholerae in intestinal tissues of the litopenaeus vannamei, the adhesion of the vibrio cholerae to the intestinal mucous membrane and the generation of virulence factors on the surface, and the disease is finally caused by the enlargement, erosion and other pathological changes of the hepatopancreas after the vibrio cholerae is further circulated to the hepatopancreas tissues through hemolymph and is propagated and generates toxin in the hepatopancreas tissues, so that the dysfunction of the hepatopancreas and the intestines is finally caused, and the morbidity and the death of the litopenaeus vannamei are caused.
(3) It is also considered by the relevant people that the nutrient substances of the feed are not completely absorbed by the intestinal tract, and the incompletely digested nutrient substances form gel substances which are mixed with the desquamated cells of the rear intestine of the prawns to form 'white feces'.
Because the occurrence mechanism of the diseases is not uniformly understood, the corresponding prevention and treatment are not greatly developed. According to the investigation, no report of using enterococcus faecalis to prevent and treat litopenaeus vannamei white feces syndrome exists.
Disclosure of Invention
The invention aims to solve the technical problem of providing a microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome as well as a preparation method and application thereof. The enterococcus faecalis of the invention can obviously inhibit vibrio cholerae (V)Vibrio cholerae) Vibrio parahaemolyticus: (VibrioParahemolyticus) Aeromonas hydrophila (b) ((b))Aeromonas hydrophila) Escherichia coli (E.coli)Escherichia coli) And the like, has broad-spectrum antibacterial activity. The microbial inoculum is used for preventing and treating the white feces disease of the litopenaeus vannamei, has remarkable prevention and treatment effect which is over 78 percent, is pollution-free and low in cost, and is convenient to popularize and use.
In order to solve the problems, the invention adopts the following technical scheme:
the invention provides a microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei, which comprises zymocyte powder of enterococcus faecalis.
Further, the fermentation liquor of the enterococcus faecalis is prepared into zymocyte powder by spray drying, and the microbial inoculum for preventing and treating the litopenaeus vannamei white feces syndrome is obtained.
The invention provides a preparation method of a microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome, which comprises the following steps: sequentially carrying out shake flask culture, primary seed culture, secondary seed culture and fermentation culture on enterococcus faecalis, and then carrying out spray drying to prepare zymocyte powder so as to prepare the microbial inoculum for preventing and treating the litopenaeus vannamei white feces syndrome.
Further, shake flask culture: performing seed shake flask culture on the enterococcus faecalis strain activated by the inclined plane, wherein the culture conditions are as follows: the rotation speed is 200-;
first-order seed culture: inoculating viable enterococcus faecalis into a primary seed culture medium according to the inoculation amount of 5-10%, and culturing at 30-37 deg.C for 10-12h to obtain a primary seed culture solution;
secondary seed culture: inoculating the primary seed culture solution into a secondary seed culture medium according to the inoculation amount of 0.06% -0.2%; culturing at 30-38 deg.C for 8-12h to obtain secondary seed culture solution;
fermentation culture, namely inoculating the secondary seed culture solution into a viable enterococcus faecalis fermentation culture medium according to the inoculation amount of 3.75-10%, and ending fermentation when the quantity of the enterococcus faecalis reaches the fermentation level;
spray drying: and (3) spray-drying the fermentation liquor to prepare fermentation bacteria powder, thus preparing the microbial inoculum for preventing and treating the white feces syndrome of the litopenaeus vannamei.
Further, the shake flask culture medium is an improved MRS culture medium, and specifically comprises the following components in parts by mass: 0.5 to 1.5 percent of peptone, 0.5 to 1.0 percent of yeast powder, 0.4 to 0.6 percent of anhydrous sodium acetate, 0.1 to 0.3 percent of dipotassium phosphate, 0.1 to 0.3 percent of diammonium citrate, 0.04 to 0.06 percent of magnesium sulfate, 0.01 to 0.03 percent of manganese sulfate, 1 to 5 percent of glucose and the balance of water, and the pH value is adjusted to be 6.4 to 6.8.
Further, the culture medium used for the first-stage seed culture comprises, by mass, 0.5-1.5% of peptone, 0.5-1.0% of yeast powder, 0.4-0.6% of anhydrous sodium acetate, 0.1-0.3% of dipotassium hydrogen phosphate, 0.1-0.3% of diammonium citrate, 0.04-0.06% of magnesium sulfate, 0.01-0.03% of manganese sulfate, 1-5% of glucose and the balance of water, wherein the pH of the culture medium is 6.4-6.8; sterilizing at 116-121 deg.C for 20-35 min.
Further, the culture medium for the second-stage seed culture comprises (by mass) peptone 0.1-0.5%, yeast extract 0.3-0.6%, glucose 0.5-1.5%, sucrose 0.5-1.5%, and K2HPO4 0.3-0.5% 、MnSO4·H2O0.01-0.03% and MgSO4·7H20.04-0.06% of O, the balance of water, and the pH value of 6.6 +/-0.2; sterilizing at 115-122 deg.C for 20-40 min;
the culture medium for fermentation comprises (by mass) peptone 0.1-0.5%, yeast extract 0.3-0.6%, glucose 0.5-1.5%, sucrose 0.5-1.5%, and K2HPO4 0.3-0.5% 、MnSO4·H2O0.01-0.03% and MgSO4·7H20.04-0.06% of O, the balance of water, and the pH value of 6.6 +/-0.2; is used after being sterilized by moist heat at 115-122 ℃ for 20-40 min.
Further, said enterococcus faecalis: (Enterococcus faecalis) CICC 20419, purchased inChina center for the preservation and management of industrial microbial strains.
Physiological and biochemical characteristics of enterococcus faecalis: enterococcus faecalis is a facultative anaerobic, gram-positive lactic acid bacterium. The streptococcus faecalis is round or oval, has a diameter of 0.5-1.0 micron, and is non-capsule and non-spore. The environmental adaptability and resistance are strong, the antibiotic-resistant coating can tolerate various antibiotics such as tetracycline, kanamycin and gentamicin, and can be extended along the chain direction; the colony is large and smooth on rich culture medium, the diameter is 1-2mm, the whole edge is full, and pigment is rare. It has low nutrient requirement, can grow in broth culture at 10 deg.C or 45 deg.C pH 9.6 or containing 6.5% NaCl, can endure 65 deg.C for 30min, and can also grow on common nutrient agar. It can utilize arginine as energy source to ferment sorbitol, and does not ferment arabinose.
The invention provides an application of a microbial inoculum for preventing litopenaeus vannamei white feces syndrome, wherein the feed is used for feeding litopenaeus vannamei in a pond without occurrence of white feces according to the proportion of 5kg-10kg of microbial inoculum for preventing and treating the litopenaeus vannamei white feces syndrome used per ton of feed.
Advantageous effects
The enterococcus faecalis has stronger stress resistance, can tolerate simulated gastric acid, simulated bile salt and high-temperature environment, can keep higher survival rate of viable bacteria, can reach more than 95 percent, and is more suitable for the requirement of feed processing.
The microbial inoculum is used for preventing and treating the white feces disease of the litopenaeus vannamei, has remarkable prevention and treatment effect which is over 80 percent, is pollution-free and low in cost, and is convenient to popularize and use.
The liquid microbial inoculum of the invention meets the requirements of mass production and use in feed factories and the processing of mixing the feed by farmers.
The liquid microbial inoculum has the characteristics of no phytotoxicity, no residue, no growth inhibition and small dosage, and provides possibility for the diversification of a medication scheme and the protection of ecological environment.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
The invention provides a microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome, enterococcus faecalis: (Enterococcus faecalis) The fermentation liquor of the method is the microbial inoculum for preventing and treating the white feces syndrome of the litopenaeus vannamei.
The invention provides a preparation method of a microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei, which comprises the following steps of sequentially carrying out shake flask culture, primary seed culture, secondary seed culture and fermentation culture on enterococcus faecalis to prepare the microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei.
The method specifically comprises the following steps: and (3) shake flask culture: performing seed shake flask culture on the strain activated by the inclined plane, wherein the shake flask culture medium is an improved MRS culture medium and comprises (by mass percent) peptone 0.5%, yeast powder 0.5%, anhydrous sodium acetate 0.5%, dipotassium phosphate 0.1%, diammonium citrate 0.1%, magnesium sulfate 0.04%, manganese sulfate 0.01%, glucose 1%, and the balance of water, and adjusting the pH value to 6.6). The culture conditions were: the rotation speed is 200r/min, the temperature is 37 ℃, and the culture is carried out for 12 h.
First-order seed culture: 100mL of primary seed culture medium is filled in a 500mL eggplant bottle, moist heat sterilization is carried out for 35min at 121 ℃, 10mL of viable enterococcus faecalis freeze-dried tube strain is inoculated in the primary seed culture medium, and the primary seed culture medium is cultured for 12h at 30 ℃; the culture medium used for first-stage seed culture is calculated according to mass fraction: peptone 0.5%, yeast powder 0.5%, anhydrous sodium acetate 0.5%, dipotassium hydrogen phosphate 0.1%, diammonium citrate 0.1%, magnesium sulfate 0.04%, manganese sulfate 0.01%, glucose 1%, and the balance of water, and adjusting the pH of the culture medium to 6.6;
secondary seed culture: 150L of secondary seed culture medium is filled in a 300L fermentation tank, moist heat sterilization is carried out for 40min at 122 ℃, and 100mL of primary seeds are inoculated in the secondary seed culture medium; culturing at 30 deg.C for 12 h; the culture medium for secondary seed culture comprises (by mass) peptone 0.2%, yeast extract 0.5%, glucose 1.0%, sucrose 1.0%, and K2HPO4 0.4% 、MnSO4·H2O0.02% and MgSO4·7H20.05 percent of O and the balance of water, and the pH value is adjusted to 6.8;
fermentation culture: at 5m3Or 30m3The expansion fermentation tank is filled with 4000L or 20m3Sterilizing enterococcus faecalis viable bacteria fermentation medium at 115 deg.C for 40min, inoculating 150L or 200L secondary seed into enterococcus faecalis viable bacteria fermentation medium, controlling tank pressure at 0.07MPa, and culturing at 30 deg.C for 36 hr; the culture medium used for fermentation comprises, by mass, 0.2% of peptone, 0.5% of yeast extract, 1.0% of glucose, 1.0% of sucrose and K2HPO4 0.4% 、MnSO4·H2O0.02% and MgSO4·7H20.05 percent of O and the balance of water; the pH value is 6.8;
ending fermentation when the quantity of the enterococcus faecalis reaches the fermentation level;
spray drying the fermentation liquor to prepare zymophyte powder; the fermentation liquor can also be directly used in prawn feed to be mixed, and has the effect of preventing and treating white feces of litopenaeus vannamei, but generally adopts the form of bacterial powder based on convenient transportation and use.
The effective viable count of enterococcus faecalis powder is not less than 1.0 × 1010cfu/g；
And tail gas treatment, namely, removing odor by adopting chemical oxidation, plasma irradiation or ultraviolet irradiation, removing ammonia nitrogen or removing hydrogen sulfide, and then discharging.
Example 2
The invention provides a microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei.e. fermentation broth of enterococcus faecalis is prepared into zymocyte powder by spray drying, and the microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei is obtained.
The invention provides a preparation method of a microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei, which comprises the following steps of sequentially carrying out shake flask culture, primary seed culture, secondary seed culture and fermentation culture on enterococcus faecalis, and then drying to prepare the microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei.
The method specifically comprises the following steps: and (3) shake flask culture: carrying out seed shake flask culture on the strain subjected to slant activation, wherein a shake flask culture medium comprises: MRS culture medium, by mass fraction, (peptone 1.0%, yeast powder 0.8%, anhydrous sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, diammonium citrate 0.2%, magnesium sulfate 0.05%, manganese sulfate 0.02%, glucose 3%, and balance water, pH value adjusted to 6.8). The culture conditions were: the rotation speed is 230r/min, the temperature is 32 ℃, and the culture is carried out for 10 hours.
First-order seed culture: 100mL of first-level seed culture medium is filled in a 500mL eggplant bottle, moist heat sterilization is carried out for 20min at 116 ℃, 10mL of viable enterococcus faecalis freeze-dried tube strain is inoculated in the first-level seed culture medium, and culture is carried out for 10h at 37 ℃; the culture medium used for first-stage seed culture is calculated according to mass fraction: peptone 1.0%, yeast powder 0.8%, anhydrous sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, diammonium citrate 0.2%, magnesium sulfate 0.05%, manganese sulfate 0.02%, glucose 3%, and the balance of water, wherein the pH of the culture medium is 6.8;
secondary seed culture: 150L of secondary seed culture medium is filled in a 300L fermentation tank, moist heat sterilization is carried out for 20min at the temperature of 115 ℃, and 300mL of primary seeds are inoculated in the secondary seed culture medium; culturing at 38 deg.C for 8 hr; the culture medium for secondary seed culture comprises (by mass) peptone 0.5%, yeast extract 0.6%, glucose 1.5%, sucrose 1.5%, and K2HPO4 0.6% 、MnSO4·H2O0.03% and MgSO4·7H20.06 percent of O and the balance of water, and the pH value is 6.6;
fermentation culture: at 5m3Or 30m3The expansion fermentation tank is filled with 4000L or 20m3Performing moist heat sterilization on an enterococcus faecalis viable bacteria fermentation culture medium at 122 ℃ for 20min, inoculating 150L or 200L of secondary seeds into the enterococcus faecalis viable bacteria fermentation culture medium, controlling the pressure of a tank to be 0.02MPa, and culturing at 38 ℃ for 24 h; the culture medium used for fermentation comprises, by mass, 0.5% of peptone, 0.6% of yeast extract, 1.5% of glucose, 1.5% of sucrose and K2HPO4 0.6% 、MnSO4·H2O0.03% and MgSO4·7H20.06 percent of O and the balance of water; the pH value is 6.6;
ending fermentation when the quantity of the enterococcus faecalis reaches the fermentation level;
spray drying the fermentation liquor to prepare zymophyte powder;
the effective viable count of enterococcus faecalis powder is not less than 1.0 × 1010cfu/g；
And tail gas treatment, namely, removing odor by adopting chemical oxidation, plasma irradiation or ultraviolet irradiation, removing ammonia nitrogen or removing hydrogen sulfide, and then discharging.
Example 3
The invention provides a microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei.e. fermentation broth of enterococcus faecalis is prepared into zymocyte powder by spray drying, and the microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei is obtained.
The invention provides a preparation method of a microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei, which comprises the following steps of sequentially carrying out shake flask culture, primary seed culture, secondary seed culture and fermentation culture on enterococcus faecalis, and then drying to prepare the microbial inoculum for preventing and treating white feces syndrome of litopenaeus vannamei.
The method specifically comprises the following steps: and (3) shake flask culture: carrying out seed shake flask culture on the strain subjected to slant activation, wherein a shake flask culture medium comprises: MRS culture medium, by mass fraction, (peptone 1.5%, yeast powder 1.0%, anhydrous sodium acetate 0.6%, dipotassium hydrogen phosphate 0.3%, diammonium citrate 0.3%, magnesium sulfate 0.06%, manganese sulfate 0.03%, glucose 5%, and balance water, pH value adjusted to 6.4). The culture conditions were: the rotating speed is 220r/min, the temperature is 35 ℃, and the culture is carried out for 11 h.
First-order seed culture: 100mL of primary seed culture medium is filled in a 500mL eggplant bottle, moist heat sterilization is carried out for 25min at 118 ℃, 10mL of viable bacteria freeze-drying tube strain of enterococcus faecalis is inoculated in the primary seed culture medium, and culture is carried out for 11h at 35 ℃; the culture medium used for first-stage seed culture is calculated according to mass fraction: peptone 1.5%, yeast powder 1.0%, anhydrous sodium acetate 0.6%, dipotassium hydrogen phosphate 0.3%, diammonium citrate 0.3%, magnesium sulfate 0.06%, manganese sulfate 0.03%, glucose 5%, and the balance of water, and adjusting the pH of the culture medium to 6.4;
secondary seed culture: 150L of secondary seed culture medium is filled in a 300L fermentation tank, moist heat sterilization is carried out for 30min at 120 ℃, and 200mL of primary seeds are inoculated in the secondary seed culture medium; culturing at 36 deg.C for 10 h; the culture medium for secondary seed culture comprises (by mass) peptone 0.1%, yeast extract 0.3%, glucose 0.5%, sucrose 0.5%, and K2HPO4 0.3% 、MnSO4·H2O 0. 01% and MgSO4·7H2O is 0.06 percent, and the pH value is adjusted to 6.6;
fermentation culture: at 5m3Or 30m3The expansion fermentation tank is filled with 4000L or 20m3Performing moist-heat sterilization on an enterococcus faecalis viable bacteria fermentation culture medium at 118 ℃ for 30min, inoculating 150L or 200L of secondary seeds into the enterococcus faecalis viable bacteria fermentation culture medium, controlling the pressure of a tank to be 0.05MPa, and culturing at 34 ℃ for 30 h; the culture medium used for fermentation comprises, by mass, 0.1% of peptone, 0.3% of yeast extract, 0.5% of glucose, 0.5% of sucrose and K2HPO4 0.3% 、MnSO4·H2O0.01% and MgSO4·7H20.06 percent of O and 6.6 of pH value of the regulator;
ending fermentation when the quantity of the enterococcus faecalis reaches the fermentation level;
spray drying the fermentation liquor to prepare zymophyte powder;
the effective viable count of enterococcus faecalis powder is not less than 1.0 × 1010cfu/g。
And tail gas treatment, namely, removing odor by adopting chemical oxidation, plasma irradiation or ultraviolet irradiation, removing ammonia nitrogen or removing hydrogen sulfide, and then discharging. Efficacy testing
(I) test of therapeutic Effect of enterococcus faecalis preparation on white feces of Litopenaeus vannamei
(1) Time and place of experiment:
the experiment is carried out in 2 open-air ponds of the Wangcun fishery in the West Qing region of Tianjin in 2018, 8 months and 6 days, and the area of each pond is 60 mu (1)#) 30 mu (2)#）。
(2) Test pond conditions:
the 2 ponds are all intensive culture shrimp ponds. Before the start of the test, white feces occurred in 2 ponds to varying degrees.
(3) The test method comprises the following steps:
1#the pond is a test pond 2#The pond is a control pond; the test starts at 1#The pond litopenaeus vannamei was dressed with the enterococcus faecalis preparation prepared in example 1 in an amount of 1kg per 100kg of feed, i.e., 10kg per ton of feed. Two for 4 days as one treatment courseThe course of treatment; 2#The pond prawns do not take any product.
1#After three days (8 months and 9 days) of the pond, the amount of the white feces on the water surface is reduced, after two days, the white feces are completely disappeared after one treatment course of mixing and taking for 8 months and 15 days, and the prawn ingests normally. And 2#The amount of white feces in the pond is increased.
(II) test of preventive effect of enterococcus faecalis preparation on white feces of Litopenaeus vannamei
(1) Time and place of experiment: the experiment is carried out in 3 open-air ponds of the Wangcun fishery in the West Qing region of Tianjin in 2018, 7 months and 6 days, and the areas of the 3 ponds are respectively 40 mu (3)#) 30 mu (4)#) 50 mu (5)#）。
(2) Test pond conditions:
the 3 ponds are all intensive culture shrimp ponds. The conditions of seedling placement, feeding, water adding, fertilizer application and the like of the three ponds are kept consistent. The first three pond shrimps were fed normally at the start of the test.
(3) The test method comprises the following steps:
random selection 3#Pond as test Pond, 4#And 5#The pond was a control pond. In a test pond, the enterococcus faecalis microbial inoculum prepared in the example 2 is continuously mixed, and the using dosage of the enterococcus faecalis microbial inoculum is 500g per 100kg of feed, namely 5kg of microbial inoculum per ton of feed. And directly feeding the feed without mixing with the lactobacillus in the control pond.
At 3 and 5 days 8 months, control group 4#And 5#White feces syndrome occurs in the pond successively; and no white feces syndrome appears in the test pond until the shrimp.
The experimental results show that the enterococcus faecalis preparation has good treatment effect and excellent prevention effect on white feces of the litopenaeus vannamei. The enterococcus faecalis of the invention can obviously inhibit vibrio cholerae: (Vibrio cholerae) Vibrio parahaemolyticus: (Vibrio Parahemolyticus) Edwardsiella tarda (Edwardsiella tarda) Aeromonas hydrophila (b) ((b))Aeromonas hydrophila) Escherichia coli (E.coli)Escherichia coli) Of pathogenic bacteriaGrowth and reproduction, and has broad-spectrum antibacterial effect. Enterococcus faecalis can reduce harmful bacteria adhesion ability and regulate intestinal microecosystem balance. And enterococcus faecalis has stronger stress resistance, can tolerate simulated gastric acid, simulated bile salt and high-temperature environment, can keep higher survival rate of viable bacteria, can reach more than 95 percent of survival rate of viable bacteria, and is more suitable for the requirement of feed processing.
The enterococcus faecalis can secrete L-type lactic acid, can convert most of nitrogen-free extracts of carbohydrates into lactic acid, and can be completely absorbed and utilized by animals. Meanwhile, enterococcus faecalis can form a biofilm in the intestines to be attached to intestinal mucosa, and can develop, grow and rapidly reproduce in the intestines to play the probiotic effect. In addition, the bacteria can decompose partial protein into amide and amino acid, and soften fiber in the feed, so that the feed has high conversion and absorption rate.
Enterococcus faecalis produces bacteriocin, organic acid or volatile fatty acid and the like to improve the environment in the intestinal tract, and the effects of competing for the nutrient components of harmful bacteria or inhibiting the growth and activity of the harmful bacteria are achieved.
Enterococcus faecalis can remarkably enhance the activity of macrophages, promote the development of immune organs, improve the immune response capability of animals, and promote the generation of antibodies and the secretion of cytokines, thereby enhancing the immunity of organisms.
Claims (6)
1. The application of the microbial inoculum for preventing and treating the litopenaeus vannamei white feces syndrome is characterized in that:
enterococcus faecalis(Enterococcus faecalis)Preparing fermentation bacteria powder from fermentation liquor of CICC 20419 by spray drying to obtain the microbial inoculum for preventing and treating the litopenaeus vannamei white feces syndrome; said enterococcus faecalis(Enterococcus faecalis)CICC 20419, purchased from China center for culture Collection of Industrial microorganisms; feeding the sick prawns according to the proportion of 5kg-10kg of microbial inoculum for preventing and treating the white feces syndrome of the litopenaeus vannamei per ton of feed.
2. The application of the microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome according to claim 1, wherein the preparation method of the microbial inoculum comprises the following steps: sequentially carrying out shake flask culture, primary seed culture, secondary seed culture and fermentation culture on enterococcus faecalis, and then carrying out spray drying to prepare a microbial inoculum for preventing and treating the litopenaeus vannamei white feces syndrome; the method comprises the following specific steps: and (3) shake flask culture: performing seed shake flask culture on the enterococcus faecalis strain activated by the inclined plane, wherein the culture conditions are as follows: the rotation speed is 200-; first-order seed culture: inoculating viable enterococcus faecalis into a primary seed culture medium according to the inoculation amount of 10%, and culturing at 30-37 deg.C for 10-12h to obtain a primary seed culture solution; secondary seed culture: inoculating the primary seed culture solution into a secondary seed culture medium according to the inoculation amount of 0.06% -0.2%, and culturing at 30-38 ℃ for 8-12h to prepare a secondary seed culture solution; fermentation culture: inoculating the second-stage seed culture solution into a viable bacteria fermentation culture medium of enterococcus faecalis according to the inoculation amount of 3.75-10%, and ending fermentation when the quantity of the enterococcus faecalis reaches the fermentation level; spray drying: and (3) spray-drying the fermentation liquor to prepare fermentation bacteria powder, thus preparing the microbial inoculum for preventing and treating the white feces syndrome of the litopenaeus vannamei.
3. The application of the microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome according to claim 2, wherein a culture medium used for shake flask culture is an improved MRS culture medium, and the culture medium comprises the following components in percentage by mass: 0.5 to 1.5 percent of peptone, 0.5 to 1.0 percent of yeast powder, 0.4 to 0.6 percent of anhydrous sodium acetate, 0.1 to 0.3 percent of dipotassium phosphate, 0.1 to 0.3 percent of diammonium citrate, 0.04 to 0.06 percent of magnesium sulfate, 0.01 to 0.03 percent of manganese sulfate, 1 to 5 percent of glucose and the balance of water, and the pH value is adjusted to be 6.4 to 6.8.
4. The application of the microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome according to claim 2, wherein the culture medium used for the first-level seed culture comprises the following components in percentage by mass: 0.5-1.5% of peptone, 0.5-1.0% of yeast powder, 0.4-0.6% of anhydrous sodium acetate, 0.1-0.3% of dipotassium phosphate, 0.1-0.3% of diammonium citrate, 0.04-0.06% of magnesium sulfate, 0.01-0.03% of manganese sulfate, 1-5% of glucose and the balance of water, and the pH value is adjusted to 6.4-6.8; sterilizing at 116-121 deg.C for 20-35 min.
5. The application of the microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome according to claim 2, wherein the culture medium used for secondary seed culture comprises the following components in percentage by mass: peptone 0.1-0.5%, yeast extract 0.3-0.6%, glucose 0.5-1.5%, sucrose 0.5-1.5%, and K2HPO4 0.3-0.5% 、MnSO4·H2O0.01-0.03% and MgSO4·7H20.04-0.06% of O, the balance of water, and the pH value of 6.6 +/-0.2; is used after being sterilized by moist heat at 115-122 ℃ for 20-40 min.
6. The application of the microbial inoculum for preventing and treating litopenaeus vannamei white feces syndrome according to claim 2 is characterized in that a culture medium used for fermentation is specifically as follows by mass fraction: peptone 0.1-0.5%, yeast extract 0.3-0.6%, glucose 0.5-1.5%, sucrose 0.5-1.5%, and K2HPO4 0.3-0.5% 、MnSO4·H2O0.01-0.03% and MgSO4·7H20.04-0.06% of O, the balance of water, and the pH value of 6.6 +/-0.2; is used after being sterilized by moist heat at 115-122 ℃ for 20-40 min.
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