CN111909868A

CN111909868A - Composite microbial inoculum for preventing and treating anorexia dead fish of grass carps as well as preparation method and application thereof

Info

Publication number: CN111909868A
Application number: CN202010725264.9A
Authority: CN
Inventors: 刘刚; 孙海远; 刘金龙
Original assignee: Tianjin Development Zone Kunhe Biotechnology Co ltd
Current assignee: Tianjin Development Zone Kunhe Biotechnology Co ltd
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2020-11-10

Abstract

The invention relates to a composite microbial inoculum for preventing and treating dead fish of grass carp anorexia and application thereof, wherein the composite microbial inoculum comprises bacillus coagulans powder and bacillus licheniformis powder; the bacillus coagulans powder is mixed with fish feed for use; the bacillus licheniformis powder is put into a pond, and the two are matched with each other, so that the grass carp feed has an obvious effect of preventing and treating anorexia and death.

Description

Composite microbial inoculum for preventing and treating anorexia dead fish of grass carps as well as preparation method and application thereof

Technical Field

The invention belongs to the field of microbial inoculant products, and particularly relates to a compound microbial inoculant for preventing and treating dead fish of grass carp anorexia as well as a preparation method and application thereof.

Background

The grass carp is lively, swims quickly, is frequently gathered to find food, and is one of four Chinese carps cultured in fresh water. Grass carps are generally favored in the middle and near-shore pasture areas of water, and are typically herbivorous fish species. Larvae, algae, grass and the like are eaten in young grass carp periods, and occasionally some 'meat foods', such as earthworms, dragonfly larvae and the like are eaten. The fish feed has delicious meat taste, convenient processing, rapid growth and wide feed sources, and becomes one of three main fish culture in northern areas of China.

Grass carp anorexia death is a frequent condition in north China aquaculture ponds, and the symptom only occurs in aquaculture ponds mainly comprising grass carps, and most of the symptoms occur when the body length of the grass carps is 15-20 cm. Anorexia and death of grass carps generally occur in 6-9 months in the high-temperature culture season. The dead grass carps have no typical symptoms and are easy to be misdiagnosed as bacterial diseases, the death tendency of the grass carps is like mild grass carp hemorrhage, but the typical symptoms of grass carp hemorrhage do not exist, and only part of diseased grass carps have black mouths.

Currently there is no good method and resistant strains to antibiotics appear after use of the antibiotics.

Current methods for treating this disease are:

1. using antidotes or sprinkled Vc

The antidote includes fruit acid products (such as citric acid and malic acid) and adsorption products (such as sodium humate and zeolite powder) for relieving anorexia and death of grass carp. And the sprinkling type Vc is used for part of farmers. However, neither method has any significant effect.

2. Using antibacterial, anti-inflammatory or disinfectant products

The currently used fishing antibiotics comprise florfenicol, sulfamonomethoxine sodium powder and the like, and the effect is not achieved after the antibiotics are orally taken, so that the number of dead grass carps is increased. Moreover, we have found that the disinfectant products increase dramatically the second day after the sprinkling and that the number of deaths does not decrease after three days.

3. Reduced feeding

A small part of breeding friends do not feed or slightly feed, so that the death number of the grass carps is reduced, but the death cannot be completely stopped. Moreover, because the disease lasts for a long time, a great number of breeding friends begin to feed again after the feeding period is reduced and the anxiety of not feeding materials is generated for one week, so that the death is increased, and the disease condition is repeated.

4. Changing water and other measures

After anorexia and death, part of culturists can choose to change water, but the situation is limited to the area where water is convenient to change, and not all the situations are suitable for water change. And the number of deaths on the day of changing water is reduced, but the number of deaths on the next day is increased in recovery.

Through continuous exploration and research of technicians of the company, the causes of death of the grass carps are deeply analyzed, and a composite microbial inoculum for preventing and treating anorexia and death of the grass carps by combining the microecological microbial inoculum is found.

Disclosure of Invention

The invention aims to provide a composite microbial inoculum for preventing and treating dead anorexia of grass carps and a preparation method and application thereof. The composite microbial inoculum comprises bacillus coagulans powder and bacillus licheniformis powder; mixing the bacillus coagulans powder with the fish feed for use; putting the bacillus licheniformis powder into a pond; the two are mutually matched, and have obvious prevention and treatment effects on anorexia and death of the grass carps.

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

a composite microbial inoculum for preventing and treating dead fish of grass carp anorexia comprises bacillus coagulans powder and bacillus licheniformis powder; the bacillus coagulans powder is mixed with fish feed for use; and putting the bacillus licheniformis powder into the pond.

As a further improvement of the above scheme:

preferably, the effective viable count of the bacillus coagulans powder is not less than 10.0 multiplied by 10⁸cfu/g, effective viable count of not less than 2.0 × 10 in Bacillus licheniformis zymocyte powder¹⁰cfu/g。

Preferably, the bacillus licheniformis in the bacillus licheniformis powder is bacillus licheniformis (b) ((b))Bacillus licheniformis) MES 816, preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the preservation date is 5 months and 8 days in 2018, and the preservation number is: CGMCC No. 15744;

the bacillus coagulans in the bacillus coagulans powder is bacillus coagulans MES847 which is preserved in China general microbiological culture collection center (CGMCC), the preservation address is No. 3 of No.1 West Lu of North Chen West Lu of the Korean-Yang district in Beijing, and the bacillus coagulans powder is preserved in the China general microbiological culture collection center of the China Committee for culture Collection of microorganisms in 2018, 5 months and 8 days, and the preservation number is CGMCC NO. 16358.

The invention also provides a preparation method of the composite microbial inoculum for preventing and treating the anorexia dead fish of the grass carps, which comprises the steps of respectively carrying out shake flask culture, primary seed culture, secondary seed culture, fermentation, concentration and spray drying on bacillus coagulans and bacillus licheniformis to prepare corresponding bacterial powder, and respectively packaging.

Wherein the preparation method of the bacillus coagulans MES847 powder is the same as the preparation method of the bacillus coagulans MES847, the microbial inoculum, the chicken feed, the preparation method and the application in the patent CN 201911381799.2;

preparation method of Bacillus licheniformis MES 816 fermentation powder is the same as the composite microbial inoculum for preventing and treating pond moss in patent application No. CN201911410610.8, and its preparation method and application are the same.

The invention also provides an application of the compound microbial inoculum for preventing and treating the anorexia dead fish of the grass carp, wherein the compound microbial inoculum is used for treating the anorexia dead fish of the grass carp:

(1) diluting the bacillus licheniformis powder with water, and spraying the diluted bacillus licheniformis powder to the whole pool, wherein 2-3 mu of the bacillus licheniformis powder is used on a water surface with the depth of 1 m per 500g of the water;

(2) the feed is fed in the first month, the daily feeding amount is 2% of the weight of the grass carp, the feeding amount is 2.5% of the weight of the grass carp from the second month, and 1kg of bacillus coagulans powder is added into each ton of the feed.

Preferably, the dilution ratio of the bacillus licheniformis powder by water is 100-500, and 200g of brown sugar is used for activating each kilogram of the bacillus licheniformis powder for 2 hours, and then the bacillus licheniformis powder is uniformly sprinkled in the whole pool.

The invention also provides an application of the compound microbial inoculum for preventing and treating the anorexia dead fish of the grass carps, wherein the compound microbial inoculum is used for preventing the anorexia dead fish of the grass carps:

(1) feeding the feed according to the daily feeding amount of 2.5 percent of the weight and the maximum feeding amount of not more than 3 percent, and adding 500g of bacillus coagulans microbial inoculum per ton of the feed;

(2) in the high-temperature season, the bacillus licheniformis powder is diluted by adding water every half month and then is sprayed to the whole pool, and 4-6 mu of the bacillus licheniformis powder is used on a water surface with the depth of 1 m for every 500 g; the medicine is used once every 10 to 15 days.

Preferably, the dilution ratio of the bacillus licheniformis powder in water is 100-500, and each kilogram of the bacillus licheniformis powder is activated by 200g of brown sugar for 2 hours and then is uniformly sprinkled in the whole pool. The significance of 2h activation is to allow bacillus to germinate rapidly and act more quickly.

The invention principle is as follows:

grass carp is mainly fed with vegetable feed, has large food intake, and can eat more than one third to two thirds of the body weight of the grass carp per day when being active. The grass carp swims fast, and is particularly fierce to rob food, so the grass carp is called as the robber grass carp by fishermen. The grass carp starts to eat, and the state of satiation can be achieved within 4 hours. Meanwhile, the digestion capability of the grass carps is very strong, and the fed grass carps can be completely digested in 4 hours. Under the condition of artificial breeding, the feeding of the compound feed reduces the feeding difficulty of the grass carps, but the grass carps still maintain strong feeding and digestion capacity, and nutrient substances are excessive if the feeding amount is controlled insignificantly. In addition, the protein content of the grass carp feed in the artificial compound feed is higher, and the grass carp fed by mixed culture materials (with higher protein) by some farmers is increased, so that the digestion and absorption burden of the grass carp is increased, the grass carp with high feed intake cannot absorb excessive protein, the resistance is reduced, diseases are easily infected, the anorexia is generated by severe people, and the grass carp can die after a long time.

According to long-term observations by the company technicians, the anorexia concrete expression of grass carps is summarized as follows: when the disease starts, the eating of the grass carps is reduced, if the feeding of the grass carps is not stopped immediately, the phenomenon is gradually aggravated, and a small amount of grass carps slowly swim on the water surface or in the water. The moribund grass carps with serious symptoms swim slowly, lie on the side, cannot keep balance of the body and die gradually.

The death rate is less in cloudy days, and the number of dead grass carps is increased in rainy days, sunny days or weather change and water change; the dead grass carps are increased by feeding the feed, and the number of the dead grass carps is reduced; increased mortality after use of disinfectants or antibiotics; there was no significant effect after using Vc splash.

The symptoms have long course and high mortality rate, the death peak generally lasts for 1-2 months, then the death amount is gradually reduced, and a part of the pond with a small amount of dead fish can continue to the pond for fish output.

After the reason and the symptoms of the anorexia of the grass carps are deeply understood, the bacillus licheniformis and the bacillus coagulans are introduced to jointly act to prevent and treat the anorexia of the grass carps.

Among them, the grass carp likes to live in a pond with fresh water quality and high transparency, and anorexia and other symptoms can be generated in the old water pond, so that the anorexia of the grass carp which is eaten too much is not easy to relieve. The bacillus licheniformis can decompose suspended substances in water, accelerate the decomposition of aged algae, and convert the aged algae into small molecules which are easily absorbed by green algae and diatom, so that the bacillus licheniformis can not only fertilize water, but also convert 'old water' into 'live water'.

The bacillus licheniformis can generate multi-active enzymes such as protease, amylase, lipase, pectinase, glucanase, cellulase and the like in the growth and metabolism process, can also generate a plurality of enzymatic factors, and has strong antagonistic action on harmful algae such as blue algae, euglena and the like. Maintaining excellent water quality, maintaining the resistance of the cultured fishes, and promoting the recovery of anorexia and dead fishes of the grass carps.

The bacillus coagulans MES847 has a wide antibacterial spectrum and has the functions of improving the cellular immunity and humoral immunity levels of organisms; can stimulate the development of immune organs of animals and improve the disease resistance of the animals; can produce active substances such as protease, lipase, amylase, xylanase and the like, and promote the digestion and absorption of feed nutrients (especially excessive fat); the generated vitamins, short-chain fatty acids, amino acids, growth promoting factors and the like can provide nutrition for animal organisms, can also promote the peristalsis of small intestines, improve the digestive function of intestinal tracts and provide favorable conditions for the growth and development of animals; the produced organic acid may function as part of the bile acid.

Therefore, the bacillus coagulans and the bacillus licheniformis act together to attack both inside and outside, so that the anorexia of the grass carps can be quickly and effectively prevented and treated.

Advantageous effects

The microbial agent has the characteristics of no phytotoxicity, no residue, no inhibition of growth and small dosage, and provides possibility for the diversification of a medication scheme and the protection of ecological environment.

The bacillus licheniformis agent has certain degradation effect on ammonia nitrogen and nitrite in aquaculture, and has regulation effect on improving water quality aging and water quality turbidity.

The bacillus coagulans can enhance the resistance and promote the absorption of redundant nutrient components (especially fat) in the feed.

The invention has low cost, quick effect and no adverse reaction for treating the disease, prevents and can promote the growth of fishes.

Detailed Description

The invention will be further illustrated with reference to specific embodiments.

Preparation method of Bacillus licheniformis MES 816 fermentation powder is the same as that of the composite microbial agent for preventing and treating pond moss, its preparation method and application in patent application No. CN 201911410610.8.

The preparation method of the bacillus coagulans MES847 bacterial powder is the same as that of the bacillus coagulans MES847 in the patent CN201911381799.2, and the bacterial agent, the chicken feed, the preparation method and the application thereof.

Example 1

A composite microbial inoculum for preventing and treating dead fish of grass carp anorexia comprises Bacillus licheniformis MES 816 bacterial powder and Bacillus coagulans MES847 bacterial powder; mixing Bacillus coagulans MES847 powder with fish feed; and (3) putting the bacillus licheniformis MES 816 bacterial powder into the pond.

The effective viable count of Bacillus coagulans powder is not less than 10.0 × 10⁸cfu/g, effective viable count of not less than 2.0 × 10 in Bacillus licheniformis zymocyte powder¹⁰cfu/g；

Respectively performing shake flask culture, primary seed culture, secondary seed culture, fermentation, concentration, spray drying to obtain corresponding bacterial powder, and respectively packaging.

Example 2

The compound microbial inoculum prepared in the embodiment 1 of the invention is used for treating dead fish with anorexia of grass carps:

the treatment experiment effect is as follows:

(1) time and place of experiment:

2 open-air ponds of a green pool-hollow fishing ground of a temple town in Tianjin City, wherein the areas of the 2 ponds are 40 mu (1 #) and 50 mu (2 #), and the average water depth is 1.8 m during the test.

(2) Test pond conditions:

the 2 ponds are all the polyculture fishponds mainly for grass carps, and the conditions of fry placing, feeding and water inflow of the two ponds are basically kept consistent. Before the test, 2 ponds are mainly euglena and assisted by green algae and diatom, and are doped with a small amount of blue algae and protozoa. Before the start of the trial, two ponds experienced unexplained grass carp death (reduced feed in the morning and at noon, and feed intake reduced to 1/3), and no obvious abnormality was found by anatomical examination. Comprehensively judging that the pond water quality is aged and excessive feed is fed in the early stage through microscopic examination of the water quality condition, so that the physique of the grass carp is reduced, and the phenomena of anorexia and death are caused. The situation is exacerbated if no action is taken.

(3) The test process comprises the following steps:

the pond # 1 is a control pond, sporadic deaths occurred in 2019, 6 and 20 months, no attention was paid, deaths increased at the beginning of 7 months, and massive deaths started in 7 and 15 months (60-70 days).

The pond was disinfected with povidone iodine starting at 7 months and 5 days, and administered orally with florfenicol. And spraying a commercially available water regulating product. The disinfectant and the antibacterial and anti-inflammatory product are continuously used for 7 months and 15 days.

The 2# pond is a test pond, sporadic death of the pond occurs at the bottom of 6 months, the death starts to increase in 7 months and 15 days, feeding is immediately reduced to 1.5% of the weight of the grass carp, 0.1% of bacillus coagulans is added into the feed, the feed is continuously fed to 8 months and 15 days, the feeding amount is 3% of the weight of the grass carp from 8 months and 16 days, and then the bacillus coagulans is added into the feed.

The bacillus licheniformis agent is diluted by 200 times for 15 days in 7 months, 25 days in 7 months and 5 days in 8 months respectively, and the bacillus licheniformis agent is uniformly sprinkled in the whole pool after 2 hours, and the product is used for 4 mu of water with the depth of 1 m per 500 g.

(4) And (3) test results:

the control pond # 1 continued until 8 months and 15 days, ending massive death, beginning to decrease until there was sporadic death of fish.

The death of the 2# experimental pond gradually decreased when the second bacillus licheniformis agent was sprinkled (25/7), and the death was basically stopped after 5/8.

Therefore, the combined preparation has obvious prevention and treatment effects on anorexia and death of the grass carps.

Example 2

The compound microbial inoculum prepared in the embodiment 1 of the invention is used for preventing dead fish with anorexia of grass carps:

the prevention experiment effect is as follows:

(1) time and place of experiment:

the method is carried out in 3 open-air ponds of a fishing farm in the new village built in Wangzhuang Zhenzhuang town of Tianjin city, wherein the areas of the 2 ponds are respectively 30 mu (1 #), 50 mu (2 #), and 40 mu (3 #), and the average water depth is 1.6 m during the test.

(2) Test pond conditions:

3 ponds are main fish ponds for being the grass carp, and the two ponds are basically consistent in fry placing, feed feeding type and water inlet condition. Before the test, 3 ponds are mainly water quality of green algae and diatoms and are doped with a small amount of cryptophyceae and blue algae and a small amount of protozoa.

(3) Test protocol:

the No.1 is a control pond, and the daily feeding amount is 3 percent of the weight of the fish; the 2# is a test pond, 3% of the weight of the fish is fed daily, 0.5% of bacillus coagulans is mixed, the bacillus licheniformis is splashed in the whole pond at intervals of 10 days to 15 days, and the using amount is 4-6 mu per 500g of water surface with the water depth of 1 m; the 3# pond is a blank group, and the daily feeding amount is 2.5 percent of the weight of the fish.

(4) The test process comprises the following steps:

the test starts in 2019, 5, 15 days, the grass carp feeding table is trained to eat on the third day after feeding, and the test starts five days later.

The 1# pond appeared to have a symptom of food cachexia in 6 months and 10 days, which is a cause of weather and was not regarded as important. Sporadic deaths occurred at 20 days 6 months, with massive deaths beginning at 30 days 6 months (20-40 deaths per day).

The pond was disinfected with povidone iodine starting at day 6 and 25 and administered orally with florfenicol. And spraying a commercially available water regulating product. The disinfectant and the antibacterial and anti-inflammatory product are continuously used for 8 months and 15 days.

Pond # 2 was a test pond that had only occasional deaths on days 7, 15, with a small number. The daily feeding amount of the pond is 3% of the weight of the fish, 0.1% of bacillus coagulans microbial inoculum is added into the feed, and the pond is continuously fed for 8 months and 15 days.

The bacillus licheniformis agent is diluted by 200 times in 5 months and 30 days, 6 months and 12 days, 6 months and 24 days, 7 months and 5 days, 7 months and 20 days, 7 months and 30 days and 8 months and 10 days respectively, the bacillus licheniformis agent is uniformly sprinkled in the whole pond after 2 hours, and the bacillus licheniformis agent is used for 4 mu of water surface with the water depth of 1 meter per 500 g.

The daily feeding amount of the 3# pond is 2.5 percent of the weight of the fish. The number of occasional deaths occurred on both 7-month and 15-month days and 8-month and 20-day days.

(5) And (3) test results:

The 3# blank pond has less death and little loss.

Therefore, the combined preparation has obvious effect of preventing anorexia and death of grass carps.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the invention, and these modifications and decorations should also be regarded as the inventive content of the present invention.

Claims

1. The composite microbial inoculum for preventing and treating the dead anorexia of the grass carps is characterized in that: the compound microbial inoculum comprises bacillus coagulans powder and bacillus licheniformis powder; the bacillus coagulans powder is mixed with fish feed for use; and putting the bacillus licheniformis powder into the pond.

2. The compound bacterial agent for preventing and treating dead anorexia of grass carps as claimed in claim 1, which is characterized in that: the effective viable count of the bacillus coagulans powder is not less than 10.0 multiplied by 10⁸cfu/g, effective viable count of not less than 2.0 × 10 in Bacillus licheniformis zymocyte powder¹⁰cfu/g。

3. The compound bacterial agent for preventing and treating dead anorexia of grass carps as claimed in claim 1, which is characterized in that: the bacillus licheniformis in the bacillus licheniformis powder is bacillus licheniformis (Bacillus licheniformis)Bacillus licheniformis) MES 816, preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the preservation date is 5 months and 8 days in 2018, and the preservation number is: CGMCC No. 15744;

4. The preparation method of the compound microbial inoculum for preventing and treating the anorexia dead fish of the grass carps as claimed in any one of claims 1 to 3, which is characterized by comprising the following steps: respectively performing shake flask culture, primary seed culture, secondary seed culture, fermentation, concentration and spray drying on the bacillus coagulans and the bacillus licheniformis to obtain corresponding bacterial powder, and respectively packaging.

5. The use of the compound microbial inoculum for preventing and treating the anorexia dead fish of the grass carps as claimed in any one of claims 1 to 3, wherein the compound microbial inoculum is used for treating the anorexia dead fish of the grass carps:

diluting the bacillus licheniformis powder with water, and spraying the diluted bacillus licheniformis powder to the whole pool, wherein 2-3 mu of the bacillus licheniformis powder is used on a water surface with the depth of 1 m per 500g of the water;

the feed is fed in the first month, the daily feeding amount is 2% of the weight of the grass carp, the feeding amount is 2.5% of the weight of the grass carp from the second month, and 1kg of bacillus coagulans powder is added into each ton of the feed.

6. The application of the composite microbial inoculum for preventing and treating the anorexia dead fish of the grass carps as claimed in any one of claim 5, wherein the dilution ratio of the bacillus licheniformis powder with water is 100-fold and 500-fold, and 200g of brown sugar is used for activating each kilogram of the bacillus licheniformis powder for 2 hours, and then the bacillus licheniformis powder is uniformly sprinkled in the whole pool.

7. The use of the compound microbial inoculum for preventing and treating the anorexia dead fish of the grass carps as claimed in any one of claims 1 to 3, wherein the compound microbial inoculum is used for preventing the anorexia dead fish of the grass carps:

(1) feeding the feed according to the daily feeding amount of 2.5 percent of the weight of the fish and not more than 3 percent at most, and adding 500g of bacillus coagulans microbial inoculum into each ton of the feed;

(2) in the high-temperature season, the bacillus licheniformis powder is diluted by adding water every half month and then is sprayed to the whole pool, and 4-6 mu of the bacillus licheniformis powder is used on a water surface with the depth of 1 m for every 500 g; is used every 10-15 days.

8. The application of the composite microbial inoculum for preventing and treating the anorexia-killed grass carp as claimed in any claim 7, wherein the dilution ratio of the bacillus licheniformis powder with water is 100-fold and 500-fold, and the whole pond is uniformly sprinkled after 2 hours.