A composite microbial preparation for aquaculture and its application in aquaculture feed

Abstract

The application discloses a compound microbial preparation for aquaculture and application of the compound microbial preparation to breeding feed, and relates to the technical field of aquaculture.The compound microbial preparation comprises Enterococcus faecium, a mold group and a Bacillus group.The contents of the Enterococcus faecium, the mold group and the Bacillus group are respectively as follows: the Enterococcus faecium is 10%-15%;the mold group is 30%-50%;and the Bacillus group is 45-55%.The technical scheme provided by the application can make the aquaculture feed have certain disease prevention and treatment effect, effectively promote the growth of aquaculture animals and improve the feed conversion rate.

Description

Technical Field

[0001] The present application relates to the field of aquaculture technology, and in particular to a composite microbial preparation for aquaculture and its application in aquaculture feed. Background Art

[0002] With the emergence of large-scale aquaculture, the breeding density continues to increase, the excrement and bait residues in the breeding water continue to increase, pathogenic microorganisms multiply in large numbers, the eutrophication of the breeding water increases, and the ecological environment is severely damaged. This leads to large-scale and frequent occurrence of various diseases and emergency situations in aquatic animals. The traditional application method is to use auxiliary additives such as antibiotics and pesticides to alleviate the above situation. However, although this method can temporarily alleviate the occurrence of diseases, it also causes aquatic animals to have problems such as high antibiotic residues.

[0003] To this end, recent research directions have gradually shifted to the use of microbial methods to achieve disease prevention and control in aquaculture, such as using the antibacterial properties of some bacteria against common pathogenic bacteria to effectively prevent the occurrence of diseases; however, aquaculture needs to consider economic benefits while ensuring disease prevention and control. How to ensure the rapid growth of farmed animals is also one of the issues that need to be considered in the breeding process. Currently, there is little related research. Summary of the Invention

[0004] The purpose of this application is to provide a composite microbial preparation for aquaculture and its application in aquaculture feed. The technical solution provided by this application enables aquaculture feed to have a certain disease prevention and control effect while effectively promoting the growth of aquaculture animals and improving feed conversion rate.

[0005] In order to achieve the above technical objectives, in a first aspect, the present application provides a composite microbial preparation for aquaculture; the composite microbial preparation comprises Enterococcus faecium, a mold group and a Bacillus group;

[0006] The contents of Enterococcus faecium, mold group and Bacillus group are respectively:

[0007] Enterococcus faecium, 10%-15%;

[0008] mold group, 30%-40%;

[0009] Bacillus group, 45-55%.

[0010] Preferably, the mold group consists of Aspergillus niger and Aspergillus oryzae.

[0011] Preferably, the Bacillus group consists of Bacillus subtilis and Bacillus coagulans;

[0012] The composite microbial preparation is composed of the following components:

[0013] Aspergillus niger, 15%-25%;

[0014] Aspergillus oryzae, 15%-25%;

[0015] Bacillus subtilis, 25%-30%;

[0016] Bacillus coagulans, 20%-25%;

[0017] Enterococcus faecium, 10%-15%.

[0018] Preferably, the Aspergillus niger has a deposit number of GDMCC3.25, and the Aspergillus oryzae has a deposit number of GDMCC3.31.

[0019] Preferably, the deposit number of the Bacillus subtilis is GDMCC1.21, and the deposit number of the Bacillus coagulans is GDMCC1.646.

[0020] Preferably, the deposit number of the Enterococcus faecium is GDMCC1.388.

[0021] Preferably, the viable count of the composite microbial preparation is not less than 998 cfu / g.

[0022] Preferably, any of the above-mentioned composite microbial preparations is mixed with aquatic feed, and the ratio of the two is 1: (45-55).

[0023] Compared with the existing technology, the beneficial effects of the present application are: through multiple compounding, the present application finally screens out the Enterococcus faecium, mold group and Bacillus group that can coexist and work together, and compound them into a composite microbial preparation; by applying the composite microbial preparation to aquaculture feed, the composite microbial preparation forms competitive inhibition of probiotics, thereby effectively reducing the disease rate and improving the survival rate of aquaculture animals; in addition, the present application can generate a large amount of digestive enzymes, such as protein digestive enzymes, starch digestive enzymes, etc. through the mold group and Bacillus group, which can effectively improve the feed conversion rate, making it easier for aquaculture animals to absorb corresponding nutrients, thereby improving the growth efficiency of aquaculture animals and improving economic benefits. DETAILED DESCRIPTION

[0024] To make the objects, features, and advantages of the present invention more readily apparent, the following detailed description of the specific embodiments of the present invention is given in conjunction with examples. Several embodiments of the present invention are provided below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present invention.

[0025] The raw materials and equipment described in the embodiments of the present invention can be purchased from any commercially available source, among which Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Bacillus coagulans, and Enterococcus faecium can all be obtained from the Guangdong Provincial Microbiological Culture Collection Center; the collection number of Aspergillus niger is GDMCC3.25, the collection number of Aspergillus oryzae is GDMCC3.31, the collection number of Bacillus subtilis is GDMCC1.21, the collection number of Bacillus coagulans is GDMCC1.646, and the collection number of Enterococcus faecium is GDMCC1.388.

[0026] The following is the relevant culture medium formula:

[0027] PDA slant medium: Take 200 g of peeled potatoes, add 1000 mL of water, boil for 20 minutes, and filter through gauze. Then, weigh 20 g of glucose and 17 g of agar and add them to the filtrate. Dose the solution to 1 L with distilled water and sterilize at 121°C for 30 minutes.

[0028] Aspergillus group expansion medium: 200 mL of potato filtrate, 2 g of glucose and 0.5 g of urea are mixed and placed in a 250 mL Erlenmeyer flask and sterilized at 121°C for 30 min.

[0029] Aspergillus group fermentation medium: peptone 10g, glucose 20g and beef extract 5g, pH 6.8.

[0030] Ordinary agar slant medium: 10g peptone, 3g beef extract powder, 5g sodium chloride, 15g agar, dilute to 1L with distilled water.

[0031] Bacillus group expansion medium: 5 g beef extract, 10 g peptone, add 5 mL water, heat until the beef extract and peptone are completely dissolved, add 500 mL water, 5 g NaCl is completely dissolved, dilute to 1 L with distilled water, adjust to pH 7.0 with NaOH, and sterilize at 121°C for 30 min.

[0032] Bacillus fermentation medium: 12 g peptone, 7 g beef extract and 5 g sodium chloride, pH 7.3.

[0033] Enterococcus faecium expansion medium: 18 g peptone, 6 g glucose, 4 g yeast extract, 0.5 g potassium dihydrogen phosphate, 0.6 g magnesium sulfate, 15 mg manganese sulfate, add 500 ml water to completely dissolve, dilute to 1 L with distilled water, adjust to pH 7.0, and sterilize at 121°C for 30 min.

[0034] Enterococcus faecium fermentation medium: 12 g peptone, 8 g corn flour, 3 g glucose, 10 g soybean meal, 2.5 g potassium dihydrogen phosphate, 3 g magnesium sulfate, add 500 ml water to completely dissolve, dilute to 1 L with distilled water, adjust to pH 7.0, and sterilize at 121°C for 30 min.

[0035] The following will further describe the preparation methods of each bacterial species and the final composite microbial preparation in this application:

[0036] (1) Preparation of Aspergillus group

[0037] Aspergillus oryzae or Aspergillus niger purchased from the Guangdong Provincial Microbial Culture Collection Center was inoculated into a PDA slant culture medium and cultured at a constant temperature of 27°C for 4-5 days. The strains on the PDA culture medium were then inoculated into an Aspergillus group expansion culture medium and cultured for another 4-5 days. Finally, the Aspergillus group expansion culture medium was inoculated into an Aspergillus fermentation culture medium at a 5% inoculum rate and cultured for 36 hours. The fermentation broth was centrifuged to obtain a centrifugal precipitate, which was freeze-dried to obtain an Aspergillus group bacterial powder.

[0038] (2) Preparation of Bacillus group

[0039] Bacillus subtilis or Bacillus coagulans purchased from the Guangdong Provincial Microbial Culture Collection Center was inoculated onto a common agar slant culture medium and cultured at 37°C for 18 hours. Subsequently, the bacteria on the common agar culture medium was inoculated onto a Bacillus group expansion culture medium and cultured at 30°C for 24 hours. Finally, the Bacillus group expansion culture medium was inoculated into a Bacillus fermentation culture medium at a 10% inoculum rate and cultured at 30°C for 12 hours. The fermentation liquid was centrifuged to obtain a centrifugal precipitate, which was freeze-dried to obtain a Bacillus group bacterial powder.

[0040] (3) Preparation of Enterococcus faecium

[0041] Enterococcus faecium purchased from Guangdong Provincial Microbial Culture Collection Center was inoculated onto ordinary agar culture and cultured at 36°C for 24 hours under anaerobic conditions. Subsequently, the bacteria on the ordinary agar culture medium were inoculated onto Enterococcus faecium expansion culture medium and cultured at 36°C for 20 hours. The culture medium was inoculated into Enterococcus faecium fermentation culture medium at a 10% inoculum volume, and then 3% mannitol and 7% milk were added. The fermentation liquid was centrifuged and precipitated, and the Enterococcus faecium powder was freeze-dried.

[0042] Example 1

[0043] A composite microbial preparation for aquaculture, consisting of the following components:

[0044] Aspergillus niger, 20%;

[0045] Aspergillus oryzae, 20%;

[0046] Bacillus subtilis, 30%;

[0047] Bacillus coagulans, 20%;

[0048] Enterococcus faecium, 10%;

[0049] The bacterial powder of each strain is weighed according to the above weight ratio and mixed evenly to obtain the composite microbial preparation described in the present application.

[0050] Example 2

[0051] A composite microbial preparation for aquaculture, consisting of the following components:

[0052] Aspergillus niger, 25%;

[0053] Aspergillus oryzae, 20%;

[0054] Bacillus subtilis, 25%;

[0055] Bacillus coagulans, 20%;

[0056] Enterococcus faecium, 10%;

[0057] The bacterial powder of each strain is weighed according to the above weight ratio and mixed evenly to obtain the composite microbial preparation described in the present application.

[0058] Example 3

[0059] A composite microbial preparation for aquaculture, consisting of the following components:

[0060] Aspergillus niger, 20%;

[0061] Aspergillus oryzae, 20%;

[0062] Bacillus subtilis, 25%;

[0063] Bacillus coagulans, 20%;

[0064] Enterococcus faecium, 15%;

[0065] The bacterial powder of each strain is weighed according to the above weight ratio and mixed evenly to obtain the composite microbial preparation described in the present application.

[0066] Example 4

[0067] A composite microbial preparation for aquaculture, consisting of the following components:

[0068] Aspergillus niger, 15%;

[0069] Aspergillus oryzae, 15%;

[0070] Bacillus subtilis, 30%;

[0071] Bacillus coagulans, 25%;

[0072] Enterococcus faecium, 15%;

[0073] The bacterial powder of each strain is weighed according to the above weight ratio and mixed evenly to obtain the composite microbial preparation described in the present application.

[0074] Example 5

[0075] A composite microbial preparation for aquaculture, consisting of the following components:

[0076] Aspergillus niger, 20%;

[0077] Aspergillus oryzae, 20%;

[0078] Bacillus subtilis, 27%;

[0079] Bacillus coagulans, 21%;

[0080] Enterococcus faecium, 12%;

[0081] The bacterial powder of each strain is weighed according to the above weight ratio and mixed evenly to obtain the composite microbial preparation described in the present application.

[0082] Example 6

[0083] A composite microbial preparation for aquaculture, consisting of the following components:

[0084] Aspergillus niger, 15%;

[0085] Aspergillus oryzae, 18%;

[0086] Bacillus subtilis, 30%;

[0087] Bacillus coagulans, 25%;

[0088] Enterococcus faecium, 12%;

[0089] The bacterial powder of each strain is weighed according to the above weight ratio and mixed evenly to obtain the composite microbial preparation described in the present application.

[0090] The present application also provides the application of the composite microbial preparation to aquaculture feed, preferably aquatic animal feed, more preferably shrimp feed;

[0091] Among them, the composite microbial preparation was not added as comparative example 1

[0092] Furthermore, the bacterial powders in Examples 1-6 were mixed according to their proportions and then mixed with commercial shrimp feed; based on the component ratio of Example 6, the ratio of the composite microbial preparation to commercial feed was 1:45 as Example 7, 1:55 as Example 8, 1:45 as Comparative Example 2, and 1:60 as Comparative Example 3.

[0093] Furthermore, the ratio of the composite microbial preparation to commercial feed in Examples 1-6 was 1:50;

[0094] In a standard indoor aquaculture pond in Huidong, Huizhou City, Guangdong Province, whiteleg shrimp were cultured. Nine ponds were selected, each 4*5m, with a water depth of 1.2-1.5m. The ponds were square, east-west oriented, with a sandy mud bottom and a pond embankment width of 2m. 20,000 shrimp fry were stocked in each pond.

[0095] According to the above embodiments, the corresponding ponds were fed respectively, and the whiteleg shrimp was fed using the satiation method, with a daily addition rate of 0.05% of the pond water volume. The initial average weight of the shrimp fry was measured to be 2.12 g / tail; the feeding period was four weeks, and the survival rate and weight gain rate of the shrimp fry were tested within four weeks. The shrimp were further tested for moisture content, crude protein content, amino acid content, and crude fat content. In addition, the adult shrimp were grouped and subjected to sensory evaluation (with a full score of 10).

[0096] Specifically, the following Table 1 is one of the test results;

[0097] Table 1

[0098] Grouping Survival rate Weight gain rate Feed coefficient Example 1 95.44 153.26±6.37 1.30±0.01 Example 2 96.39 158.30±5.18 1.28±0.01 Example 3 94.68 150.32±6.18 1.30±0.02 Example 4 95.21 154.36±5.44 1.29±1.02 Example 5 97.88 160.28±6.87 1.24±0.02 Example 6 99.02 176.56±5.63 1.12±0.02 Example 7 98.02 170.66±6.62 1.20±0.01 Example 8 98.23 171.25±6.57 1.22±0.02 Comparative Example 1 87.49 105.69±7.25 1.49±0.01 Comparative Example 2 96.22 162.78±5.97 1.27±0.03 Comparative Example 3 97.15 164.01±6.33 1.25±0.01

[0099] Examples 6-8 and control examples 1-3 were sampled for moisture content, crude protein content, amino acid content, and crude fat content. The specific results are as follows:

[0100] Specifically, Table 2 is one of the test results;

[0101] Table 2

[0102]

[0103]

[0104] Further comprehensive analysis of the above results, comparing Examples 1-6 with Control Example 1, shows that after using the aquaculture feed added with the composite microbial preparation, the survival rate and weight gain rate of the shrimp are significantly improved. Among them, Example 6 is the best, with the survival rate increased by more than ten percentage points compared with Control Example 1; in addition, from the analysis of the feed coefficient, the use of the composite microbial preparation also effectively reduces the feed coefficient, thereby making the aquatic feed have a higher conversion efficiency and improving economic benefits.

[0105] Furthermore, from the quality test results of the shrimp, the crude protein content and amino acid content of the shrimp in Example 6 were significantly higher than those in Control Example 1, and the moisture content was controlled within a more optimal range, and the crude fat content was also lower.

[0106] Further analysis of Examples 6-8 and Control Examples 2-3 shows that the ratio of the addition amount of the composite microbial preparation to the aquaculture feed also has a certain impact on the final shrimp quality. When the addition amount of the composite microbial preparation is too high or too low, the optimal effect of use cannot be achieved.

[0107] In summary, the composite microbial preparation provided in the present application is used for aquaculture feed, and through the competitive inhibition of probiotics, it effectively alleviates the occurrence of aquatic diseases, thereby improving the survival rate of aquatic animals; at the same time, by rationally compounding various bacterial species and corresponding proportions, while ensuring the mutual beneficial symbiosis of various bacterial species, the role of each bacterial species is maximized, and thus, while ensuring the effective inhibition of pathogenic bacteria, other beneficial bacteria that produce digestive enzymes can function normally, thereby having the dual functions of preventing diseases and promoting the growth of aquatic animals.

[0108] It should be noted that, unless there is any conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0109] The above description is only a preferred embodiment of the present application and does not constitute any form of limitation to the present application. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present application fall within the scope of the technical solution of the present application.

Claims

1. An application of a composite microbial preparation for aquaculture in the preparation of shrimp aquaculture feed, characterized in that: The composite microbial preparation is composed of the following components by weight: Aspergillus niger, 15%; Aspergillus oryzae, 18%; Bacillus subtilis, 30%; Bacillus coagulans, 25%; Enterococcus faecium, 12%; The Aspergillus niger has a deposit number of GDMCC3.25, and the Aspergillus oryzae has a deposit number of GDMCC3.31; The deposit number of the Bacillus subtilis is GDMCC1.21, and the deposit number of the Bacillus coagulans is GDMCC1.646; The deposit number of the Enterococcus faecium is GDMCC1.388; The application method is to mix the composite microbial preparation with aquatic feed, with the ratio of the two being 1:

50.

2. The use according to claim 1, characterized in that: The bacterial viability of the composite microbial preparation is not less than 998 cfu / g.

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