A kind of probiotic fermented traditional Chinese medicine for fishery and its preparation and application

Abstract

The present invention discloses a probiotic fermented Chinese medicine for fishery use, and its preparation and application. A probiotic fermented Chinese medicine preparation for fishery use is characterized in that: the Chinese medicinal ingredients in the probiotic fermented Chinese medicine preparation are honeysuckle, dandelion, and licorice; and the probiotics in the probiotic fermented Chinese medicine preparation are Bacillus inaquosorum strain L-14, Bacillus tequilensis strain 6-9, and Enterococcus faecium strain RY2. The probiotic fermented Chinese medicine provided by the present invention can be added to feed to prevent and control bacterial hemorrhagic disease in fish and promote fish growth.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation feed additives, and in particular to a probiotic fermentation traditional Chinese medicine for fishery use, and the preparation and application thereof. Background Art

[0002] my country is the world's largest producer and consumer of freshwater fish, making the sustainable development of freshwater fisheries crucial. Due to the homogeneous water environment, large fish populations, and the highly contagious nature of diseases, the extensive use of antibiotics poses widespread risks, making prevention more important than treatment.

[0003] Traditional Chinese medicine, with its natural ingredients, safe efficacy, and numerous functions, can play a role in preventing diseases in fishery production. Probiotics, on the other hand, have the functions of regulating intestinal function, maintaining the ecological balance of intestinal flora, enhancing digestion and immunity, and promoting growth, and are important components of feed additives. The preparation of fishery feed additives with probiotic fermentation of traditional Chinese medicine combines the advantages of both. Natural traditional Chinese medicine is used as the matrix, and a variety of probiotics are added for fermentation. On the one hand, the effective active ingredients of traditional Chinese medicine are fully exerted. On the other hand, the various enzymes and antibacterial secondary metabolites produced help digestion, absorption, and metabolism of nutrients in fishery feed. This can not only effectively prevent and control the occurrence of diseases, but also promote the growth and weight gain of fish, improve feed conversion rate, and maintain the homeostasis of fish intestinal microbial flora. It plays a key role in the fishery transformation of reducing and replacing antibiotics. Summary of the Invention

[0004] The present invention aims to provide a probiotic fermented Chinese medicine for fishery use and its preparation and application.

[0005] In a first aspect, the present invention claims protection for a probiotic fermented Chinese medicinal preparation for fishery use.

[0006] The Chinese medicinal ingredients in the probiotic fermented Chinese medicinal preparation for fishery claimed by the present invention are honeysuckle, dandelion and licorice, and the probiotics are Enterococcus faecium strain RY2, Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14.

[0007] The Enterococcus faecium strain RY2 is numbered ACCC 62687 at the Agricultural Microbiology Center of the China Culture Collection Administration;

[0008] The Bacillus tequilensis strain 6-9 is numbered ACCC 62688 at the Agricultural Microbiology Center of the China Culture Collection Administration;

[0009] The Bacillus inaquosorum strain L-14 is numbered ACCC 62689 in the Agricultural Microbiology Center of the China Culture Collection Administration.

[0010] Honeysuckle is the dried buds or newly opened flowers of Lonicera japonica Thunb., a plant of the genus Lonicera in the family Caprifoliaceae. It is sweet and cold in nature and enters the lung and stomach meridians. It is used to treat exogenous wind-heat or fever caused by febrile diseases, heatstroke, dysentery with bloody heat toxins, carbuncles, furuncles, sore throat, and various infectious diseases.

[0011] Dandelion, also known as yellow flower dandelion, dandelion, and flower flower, is the dried whole herb of Taraxacum mongolicum Hand.-Mazz., Taraxacum borealisinense Kitag., or several species of the same genus, all belonging to the Asteraceae family. It has a bitter, sweet, and cold flavor. It enters the Liver and Stomach meridians. It is primarily used to treat boils, breast abscesses, scrofula, conjunctivitis, sore throat, lung abscesses, intestinal abscesses, damp-heat jaundice, and painful stranguria caused by heat.

[0012] Licorice is the dried root and rhizome of the legume plants Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L. It has a sweet flavor and a neutral nature. It enters the Heart, Lung, Spleen, and Stomach meridians. It tonifies the spleen and replenishes Qi, clears away heat and detoxifies, eliminates phlegm and relieves cough, relieves pain, and harmonizes various medicinal properties. It is commonly used for spleen and stomach deficiency, fatigue, palpitations, shortness of breath, cough with sputum, acute pain in the abdomen and limbs, carbuncles, sores, and relieves drug toxicity and potency.

[0013] Furthermore, the probiotic fermented Chinese medicine preparation for fishery use may also contain fermentation auxiliary materials, and the fermentation auxiliary materials include corn flour, bran and soybean meal.

[0014] Furthermore, the probiotic fermented Chinese medicine preparation for fishery use can be obtained by inoculating the probiotic into a Chinese medicine fermentation medium for fermentation and culturing; the Chinese medicine fermentation medium contains the Chinese medicine ingredients and the fermentation auxiliary materials.

[0015] In the traditional Chinese medicine fermentation medium, the weight ratio of honeysuckle, dandelion and liquorice as the traditional Chinese medicine ingredients can be 4:2:1.

[0016] In the traditional Chinese medicine fermentation medium, the weight ratio of corn flour, bran and soybean meal as the fermentation auxiliary materials can be 10:4:1;

[0017] In the traditional Chinese medicine fermentation medium, the weight ratio of the traditional Chinese medicine components to the fermentation auxiliary materials can be 3:7.

[0018] Among them, the probiotics can be inoculated into the traditional Chinese medicine fermentation medium by first mixing the Bacillus inaquosorum strain L-14 and the Bacillus tequilensis strain 6-9 and inoculating them into the traditional Chinese medicine fermentation medium, and then inoculating the Enterococcus faecium strain RY2 after cultivation.

[0019] Furthermore, the ratio of the number of live bacteria of the Bacillus inaquosorum strain L-14, the Bacillus tequilensis strain 6-9 and the Enterococcus faecium strain RY2 inoculated into the traditional Chinese medicine fermentation medium is 1:1:2.

[0020] Furthermore, the bacterial amounts of the Bacillus inaquosorum strain L-14, the Bacillus tequilensis strain 6-9, and the Enterococcus faecium strain RY2 inoculated into each gram of the traditional Chinese medicine fermentation medium are 5×10 6 cfu, 5×10 6 cfu and 1 × 10 7 cfu.

[0021] In a specific embodiment of the present invention, the probiotic fermentation Chinese medicine preparation for fishery use can be prepared according to a method comprising the following steps:

[0022] (A1) Drying honeysuckle, dandelion, and liquorice (e.g., drying at 60° C.), crushing the mixture, and mixing the mixture in a weight ratio of 4:2:1 to obtain a mixed Chinese medicine powder.

[0023] In a specific embodiment of the present invention, after the medicinal materials are crushed, the step of passing through a 40-mesh sieve is further included.

[0024] (A2) The mixed Chinese medicine powder in (A1) is mixed with a fermentation auxiliary material in a weight ratio of 3:7 to obtain a Chinese medicine fermentation medium; the fermentation auxiliary material is formed by mixing corn flour, bran and soybean meal in a weight ratio of 10:4:1.

[0025] In this step, the components of the Chinese medicine fermentation medium are added to a triangular flask according to the proportion, sterilized at 121°C for 30 minutes to effectively kill harmful bacteria in the Chinese medicine fermentation medium, and then cooled to 32°C.

[0026] (A3) The concentration is 10 8 cfu / mL of the fermentation culture broth (primary seed liquid) of the Bacillus inaquosorum strain L-14 and the fermentation culture broth (primary seed liquid) of the Bacillus tequilensis strain 6-9 were mixed in a volume ratio of 1:1 to obtain a Bacillus mixed bacterial liquid (secondary seed).

[0027] (A4) The mixed bacterial solution of Bacillus spp. (A3) was added to a total bacterial count of 1×10 7 cfu / g (per g refers to per gram of the Chinese medicine fermentation medium) is added to the Chinese medicine fermentation medium (A2), the water content is adjusted to 50% (weight percentage), and the culture is allowed to ferment at 32°C for 3-5 days (e.g., 5 days) to obtain a Bacillus Chinese medicine fermentation culture; then, the bacterial count is 1×10 7 cfu / g (per g refers to per gram of the Chinese medicine fermentation medium) was added to the Bacillus Chinese medicine fermentation culture at a concentration of 10 8 cfu / mL of the fermentation culture broth (primary seed liquid) of the Enterococcus faecium strain RY2 is stirred evenly, allowed to stand at 32°C for 2 days for adsorption, and then air-dried to obtain the probiotic fermentation Chinese medicine preparation for fishery use.

[0028] In steps (A3) and (A4), the primary seed solution of each strain is prepared by inoculating the corresponding strain into the corresponding culture medium and performing single strain enrichment culture to 10 8 cfu / mL was obtained.

[0029] The culture medium for the three strains is as follows:

[0030] Bacillus inaquosorum strain L-14, Bacillus equilensis strain 6-9, and Enterococcus faecium strain RY2 were inoculated into a fermentation liquid medium with the following formula: glucose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4 2 g / L, NaCl 0.5 g / L, and sterilized by autoclaving at 121°C for 15 min. The inoculum was inoculated at a volume ratio of 1:100 and cultured at 37°C for 18-24 h to obtain a bacterial concentration of 10 8 cfu / mL of the first-level seed solution.

[0031] In a second aspect, the present invention claims a method for preparing a probiotic fermented Chinese medicine preparation for fishery use.

[0032] The method for preparing a probiotic fermented Chinese medicine preparation for fishery use claimed in the present invention may include steps (A1) to (A4) described above.

[0033] In a third aspect, the present invention claims protection for fish feed containing the fishery probiotic fermented Chinese medicine preparation described in the first aspect above.

[0034] Furthermore, the fish feed of the probiotic fermented Chinese medicine preparation for fishery is obtained by adding 1% of the total weight of the probiotic fermented Chinese medicine preparation for fishery to fish feed.

[0035] In a fourth aspect, the present invention claims protection for any of the following products or applications:

[0036] (B1) A complete set of products for preparing a probiotic fermented Chinese medicine preparation for fishery use, comprising: honeysuckle, dandelion, liquorice, corn flour, bran, soybean meal, Bacillus inaquosorum strain L-14 described in the first aspect above, Bacillus tequilensis strain 6-9 described in the first aspect above, and Enterococcus faecium strain RY2 described in the first aspect above;

[0037] (B2) A complete product set for preparing a probiotic fermented Chinese medicine preparation for fishery use, comprising: the Chinese medicine fermentation medium described in the first aspect above, the Bacillus inaquosorum strain L-14 described in the first aspect above, the Bacillus tequilensis strain 6-9 described in the first aspect above, and the Enterococcus faecium strain RY2 described in the first aspect above;

[0038] (B3) Use of the probiotic fermented Chinese medicinal preparation for fishery use described in the first aspect, the fish feed described in the third aspect, or the complete set of products described in (B1) or (B2) in the preparation of a product for preventing bacterial hemorrhagic disease in fish;

[0039] (B4) Use of the probiotic fermented Chinese medicine preparation for fishery use described in the first aspect above, or the fish feed described in the third aspect above, or the set of products described in (B1) or (B2) in the preparation of products for promoting fish growth.

[0040] In the above aspects, the fish bacterial hemorrhagic disease is fish bacterial hemorrhagic disease caused by Aeromonas hydrophila.

[0041] In the above aspects, the fish bacterial hemorrhagic disease may be acute fish bacterial hemorrhagic disease or chronic fish bacterial hemorrhagic disease.

[0042] In a specific embodiment of the present invention, the prevention of fish bacterial hemorrhagic disease is specifically embodied by increasing the survival rate (reducing the mortality rate).

[0043] In a specific embodiment of the present invention, the promoting fish growth is specifically embodied as promoting fish weight gain.

[0044] In all the above aspects, the fish may be freshwater fish.

[0045] In a specific embodiment of the present invention, the fish in the above aspects is grass carp.

[0046] The technical solution provided by the present invention has the following advantages:

[0047] (1) The probiotic fermented Chinese medicinal feed provided by the present invention uses probiotic fermented Chinese medicinal materials and commonly used fishery feed as raw materials, which fully guarantees the safety of the feed raw materials. The selected Chinese medicinal materials have the activities of clearing away heat and detoxifying, and antibacterial and tonifying the middle part. The integration of the two plays the role of regulating immune balance, antibacterial and anti-inflammatory, and promoting growth and weight gain.

[0048] (2) The probiotic fermented Chinese medicinal feed provided by the present invention is prepared by drying and crushing the Chinese medicinal materials and then fermenting them with enriched mixed probiotics, thereby shortening the fermentation period of the Chinese medicinal materials while fully stimulating the active ingredients of the Chinese medicinal materials.

[0049] (3) The probiotic-fermented Chinese medicinal feed provided by the present invention can improve feed utilization, increase feed intake, and promote nutrient absorption in animals. Digestive enzymes such as protease and cellulase produced by probiotics help break down protein and carbohydrate macromolecules in feed, reducing the digestive burden on farmed animals and improving feed conversion efficiency.

[0050] (4) The probiotic fermented Chinese medicinal feed provided by the present invention can regulate the balance of fish intestinal flora and improve the immune ability and disease resistance of the animal body. Bacillus can produce antibacterial and antibacterial active substances, effectively inhibiting the growth of Aeromonas hydrophila, thereby reducing the harm of bacterial pathogens infection; and Enterococcus faecium adapts to the living environment of fish intestines, and its large-scale colonization and proliferation can promote the absorption and metabolism of feed nutrients. The synergistic effect of the two can effectively improve the disease resistance and immunity of animals, not only can it prevent and control grass carp bacterial hemorrhagic disease caused by Aeromonas hydrophila, significantly reduce the mortality rate of grass carp (50%), increase the protection rate to 33%, alleviate the surface and abdominal symptoms of grass carp, but also promote the growth of grass carp, with an average weight gain of 12.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The diagram shows the antagonism of Bacillus tequilensis strain 6-9 (A) and Bacillus inaquosorum strain L-14 (B) on an Aeromonas hydrophila plate.

[0052] Figure 2 The colony morphology of Bacillus tequilensis strain 6-9 (A) and Bacillus inaquosorum strain L-14 (B) of the present invention is shown.

[0053] Figure 3 The phylogenetic tree is constructed based on the 16S rRNA gene sequences of the strains 6-9 and L-14 of the present invention and the type strains of the genus Bacillus.

[0054] Figure 4 The growth of Bacillus tequilensis strain 6-9 (A) and Bacillus inaquosorum strain L-14 (B) of the present invention after culturing at 30° C. and different pH conditions for 48 hours.

[0055] Figure 5 The bile salt tolerance of Bacillus tequilensis strain 6-9 (A) and Bacillus inaquosorum strain L-14 (B) of the present invention at 30° C. and pH 7.0.

[0056] Figure 6 It is the protease activity of the fermentation broth of the Bacillus tequilensis strain 6-9 and the Bacillus inaquosorum strain L-14 of the present invention cultured at 30° C. and pH 7.0 for 48 hours.

[0057] Figure 7 The cellulase activity of the fermentation broth of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14 of the present invention cultured at 30° C. and pH 7.0 for 48 hours with CMC-Na as a substrate is measured.

[0058] Figure 8The cellulase activity of the fermentation broth of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14 of the present invention cultured at 30° C. and pH 7.0 for 48 hours using filter paper as a substrate. DETAILED DESCRIPTION

[0059] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0060] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0061] The Enterococcus faecium strain RY2 in the following examples has been collected in the Agricultural Microorganism Center of the China Culture Collection of Microorganisms, also known as the Agricultural Culture Collection of China (ACCC, address: No. 12, Zhongguancun South Street, Haidian District, Beijing, Institute of Agricultural Resources and Agricultural Zoning, Chinese Academy of Agricultural Sciences, Postal Code 100081) before the filing date of this application. The strain number is ACCC 62687, and the collection date is May 17, 2022. From the date of collection, the public can obtain the strain from the Agricultural Microorganism Center of the China Culture Collection of Microorganisms. ACCC has a dedicated website at: http: / / www.accc.org.cn, and the public can order strains directly online. The partial sequence of the 16S rRNA gene of strain RY2 is shown in SEQ ID No. 3.

[0062] The Bacillus tequilensis strains 6-9 in the following examples have been collected at the Agricultural Microorganism Center of the China Culture Collection of Microorganisms, also known as the Agricultural Culture Collection of China (ACCC, address: 12 Zhongguancun South Street, Haidian District, Beijing, Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences, zip code 100081) before the filing date of this application. The strain number is ACCC 62688, and the collection date is May 17, 2022. From the date of collection, the public can obtain the strain from the Agricultural Microorganism Center of the China Culture Collection of Microorganisms. ACCC has a dedicated website at http: / / www.accc.org.cn, and the public can order strains directly online. The partial sequence of the 16S rRNA gene of strain 6-9 is shown in SEQ ID No. 1.

[0063] The Bacillus inaquosorum strain L-14 in the following examples has been collected by the Agricultural Microorganism Center of the China Culture Collection of Microorganisms, also known as the Agricultural Culture Collection of China (ACCC, address: No. 12, Zhongguancun South Street, Haidian District, Beijing, Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences, Postal Code 100081) before the filing date of this application. The strain number is ACCC 62689, and the collection date is May 17, 2022. From the date of collection, the public can obtain the strain from the Agricultural Microorganism Center of the China Culture Collection of Microorganisms. ACCC has a dedicated website at http: / / www.accc.org.cn, and the public can order strains directly online. The partial sequence of the 16S rRNA gene of strain L-14 is shown in SEQ ID No. 2.

[0064] Example 1. Isolation and Identification of Bacillus tequilensis Strain 6-9 and Bacillus inaquosorum Strain L-14

[0065] 1. Isolation of strains

[0066] Healthy grass carp (freshwater fish) purchased from the fish pond in Changping District, Beijing, and grouper (marine fish) purchased from the seafood market in Ledong City, Hainan Province, were anesthetized with MS-222. The abdominal wall muscles were cut open along the midline of the abdomen from the anus and the side wall muscles were cut off to expose the abdominal cavity. The abdominal cavity was flushed with sterile PBS buffer. The intestine was cut and placed in a 12 mL sterile shaking tube. 5 mL of PBS buffer was added and the intestine was fully minced with sterile surgical scissors. The mixture was shaken at 37°C and 200 rpm for 30 minutes. After the mixture was allowed to stand for 2 minutes, 1 mL of the supernatant was taken and diluted 10-fold to 10 -2 -10 -4 Select three dilution gradients and evenly spread 20 μL of each dilution onto LB solid medium, performing three replicates for each dilution. Incubate the cells in an inverted position at 37°C for 24-48 hours. Select colonies of varying morphology and obtain 50 and 23 strains from the intestines of healthy grass carp and grouper, respectively. After three plate passages, a genetically stable monoclonal strain was obtained and stored at -80°C.

[0067] 2. Screening of antagonistic bacteria

[0068] 1. Pre-screening by plate antagonism method

[0069] Aeromonas hydrophila was cultured in LB liquid medium at 37°C and 200 rpm for 24 h, and diluted to 10 7 cfu / mL. 20 μL of the diluted bacterial solution was evenly spread on LB solid medium. A single colony of the pure test bacteria was selected and two short horizontal lines were drawn on the plate. Three groups were drawn on one plate, with two parallel groups. The plates were incubated in a 37°C incubator for 18-24 hours. 37 / 50 strains isolated from the intestines of grass carp and 7 / 23 strains isolated from the intestines of grouper produced obvious inhibition zones.

[0070] 2. Oxford Cup Method Rescreening

[0071] Take a 9cm diameter sterile petri dish with sterilized 1.5% agar as the bottom, place 4 sterile Oxford cups on the solidified agar with tweezers, cool the sterilized LB solid medium to 50℃ and add Aeromonas hydrophila culture solution to 10 5 cfu / mL, and then pour it into the culture dish with the Oxford cup. After the culture medium solidifies, remove the Oxford cup and add 100 μL of the test strain solution (10 7cfu / mL), with 50μg / mL chloramphenicol as the control. The culture dish was placed in a refrigerator at 4°C for 1 hour to allow the active substance to fully diffuse in the medium, and then the culture dish was cultured at 37°C for 18-24 hours. The experiment was set up in 3 parallels. The antibacterial activity of the strain was evaluated by measuring the diameter of the inhibition zone and subtracting the diameter of the Oxford cup, in mm. The results showed that 12 strains of bacteria had an inhibition zone of more than 5mm. Figure 1 As shown, compared with the inhibition zone of 17.4±0.5 mm of chloramphenicol, the inhibition zone diameters of strains No. 6-9 and L-14 were 5.3±0.5 mm and 9.3±0.2 mm, respectively, which were 30.5% and 53.4% ​​of the diameter of the antibiotic inhibition zone, respectively.

[0072] 3. Morphological Observation and 16S rRNA Identification of Strains 6-9 and L-14

[0073] Take strains 6-9 and L-14 stored at -80℃ and culture them on LB solid medium at 37℃ for 2 days. Observe the colony morphology. The colonies are dirty white, rough and irregular, with ridges and wrinkles. Figure 2 shown.

[0074] Genomic DNA from strains 6-9 and L-14 was extracted, respectively, and PCR amplification was performed using the bacterial 16S rRNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction system and PCR procedure are shown in Table 1 and Table 2, respectively. The purity and size of the PCR amplification products were checked by 1.2% agarose gel electrophoresis. PCR products of the correct length were sent to Shanghai Sangon for sequencing.

[0075] Table 1. PCR reaction system

[0076] Reagents Dosage (μL) 2×Taqmix 12.5 Primer 27F (10 pmol / μL) 1.0 Primer 1492R (10 pmol / μL) 1.0 DNA template 0.5 <![CDATA[ddH20]]> 10.0 Total volume 25.0

[0077] Table 2. PCR amplification program

[0078]

[0079] The partial sequence of the 16S rRNA gene of strain 6-9 is shown in SEQ ID No. 1, and the partial sequence of the 16S rRNA gene of strain L-14 is shown in SEQ ID No. 2. According to the BLAST results, the similarity between the 16S rRNA gene sequence of strain 6-9 and the model strain KJ847721 of Bacillus teslaensis is 99.66%; the similarity between the 16S rRNA gene sequence of strain L-14 and the model strain KCTC13429 of Bacillus inaquosorum is 99.86%. The phylogenetic analysis of the 16S rRNA gene sequences of strains L-14 and 6-9 and the model strains of the genus Bacillus was performed using the Neighbor-Joining Tree algorithm using MEGA X software. Figure 3 As shown, the constructed phylogenetic tree showed that strain 6-9 was listed in the same branch as the type strain KJ847721 of Bacillus tequilensis, and L-14 was listed in the same branch as the type strain KCTC13429 of Bacillus inaquosorum.

[0080] Based on the colony morphology and 16S rRNA gene sequence, it can be determined that strain 6-9 belongs to Bacillus tequilensis; L-14 belongs to Bacillus inaquosorum, and they were named Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14, respectively. They were deposited in the Agricultural Microbiology Center of China Culture Collection Administration, No. 12 Zhongguancun South Street, Haidian District, Beijing, Institute of Agricultural Resources and Agricultural Zoning, Chinese Academy of Agricultural Sciences on May 17, 2022, with the accession numbers ACCC 62688 and ACCC 62689, respectively.

[0081] IV. Acid, Alkali, and Bile Salt Tolerance of Strains 6-9 and L-14

[0082] Single colonies of strains 6-9 and L-14 were picked up and placed in 1 mL of LB liquid medium. The culture tubes were shaken at 37°C and 200 rpm for 24-36 h. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded and the cells were resuspended in sterile PBS. The OD was measured using a microplate reader. 600 Then adjust the initial concentration of the bacterial solution to OD 600 Value 0.15±0.01.

[0083] First, add 190 μL of LB liquid medium with different pH values ​​(3.0, 5.0, 6.0, 7.0, 8.0, and 9.0) to a sterile 96-well bacterial culture plate, then add 10 μL of L-14 or 6-9 bacterial solution, and culture at 37°C for 48 h. OD was measured using a microplate reader. 600 The pH value was 7.0 as the control, and each treatment was repeated three times. Figure 4 As shown, strains 6-9 and L-14 grew well at pH 3.0-9.0, and their optimal growth conditions were pH 5.0-8.0 and pH 6.0-8.0, respectively.

[0084] First, 190 μL of LB liquid medium containing different concentrations (0.03%, 0.05%, 0.1%, 0.2%, 0.3%, % represents g / 100 mL) of pig bile salt (Solebol G-8310) was added to a sterile 96-well bacterial culture plate, and then 10 μL of strain 6-9 or L-14 bacterial solution was added. The plates were cultured at 37°C for 48 h, and the OD was measured using a microplate reader. 600 The values ​​were calculated, with no bile salt as the control, and three replicates were used for each treatment. Figure 5 As shown, strains 6-9 and L-14 have a certain tolerance to bile salts and can grow at bile salt concentrations of 0.03% and 0.05%.

[0085] 5. Protease and cellulase activities of fermentation broths of strains 6-9 and L-14

[0086] Fermentation medium: glucose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4 2 g / L, NaCl 0.5 g / L, distilled water to 1 L, autoclave at 121°C for 15 min for later use.

[0087] Strains 6-9 and L-14 were cultured in LB liquid medium with shaking at 37°C and 200 rpm for 24 h, inoculated into fermentation medium at a 2% (volume percentage) inoculation rate, cultured at 37°C and 200 rpm for 48 h, and centrifuged at 8000 rpm for 15 min at 4°C. The supernatant was used as the crude enzyme solution.

[0088] Reagents were prepared according to the national standards SB / T 10317-1999, "Protease Activity Assay," and QB2583-2003, "Cellulase Preparations," and the protease and cellulase activities in the fermentation broths of strains L-14 and 6-9 were measured and calculated. As shown in Table 3, the protease and cellulase activities of strain L-14 were higher than those of strain 6-9. Figure 6It is the protease activity of the fermentation broth of the Bacillus tequilensis strain 6-9 and the Bacillus inaquosorum strain L-14 of the present invention cultured at 30° C. and pH 7.0 for 48 hours. Figure 7 The cellulase activity of the fermentation broth of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14 of the present invention, which is cultured at 30° C. and pH 7.0 for 48 hours, using CMC-Na as a substrate. Figure 8 The cellulase activity of the fermentation broth of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14 of the present invention cultured at 30° C. and pH 7.0 for 48 hours using filter paper as a substrate.

[0089] Table 3. Protease and cellulase activities of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14

[0090]

[0091] Example 2: Preparation of probiotic fermented Chinese medicinal feed for fishery

[0092] 1. Preparation of probiotic fermented Chinese medicine for fishery

[0093] (1) Preparation of mixed Chinese herbal powder: drying honeysuckle, dandelion and liquorice at 60°C, crushing and passing through a 40-mesh sieve, weighing each component by weight, stirring evenly to obtain mixed Chinese herbal powder for later use; the weight proportions of each Chinese herbal ingredient are: 4 parts of honeysuckle, 2 parts of dandelion and 1 part of liquorice.

[0094] (2) Preparation of Chinese medicine fermentation medium: The mixed Chinese medicine powder and fermentation auxiliary materials are mixed in a ratio of 3:7 (weight ratio). The fermentation auxiliary materials are obtained by mixing 10 parts by weight of corn flour, 4 parts by weight of bran, and 1 part by weight of soybean meal.

[0095] Add the components of the Chinese medicine fermentation medium into a triangular flask according to the proportion, sterilize at 121°C for 30 minutes to effectively kill harmful bacteria in the Chinese medicine fermentation medium, and then cool to 32°C.

[0096] (3) Preparing a single-species primary seed solution of probiotics, Bacillus inaquosorum strain L-14, Bacillus tequilensis strain 6-9, and Enterococcus faecium strain RY2 were inoculated into a fermentation liquid culture medium, respectively, to obtain a single-species primary seed solution of various probiotics.

[0097] The fermentation liquid medium formula is as follows: glucose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, MgSO4 2 g / L, NaCl 0.5 g / L, and autoclaved at 121 °C for 15 min.

[0098] The inoculum volume ratio was 1:100, and the culture was carried out at 37℃ for 16-24h to obtain a bacterial concentration of 10 7 cfu / mL of the first-level seed solution.

[0099] (4) preparing a secondary seed solution of multiple strains of Bacillus, and mixing the single strain primary seed solution (10 7 cfu / mL) were mixed at a volume ratio of 1:1 to obtain a Bacillus multi-species mixed secondary seed liquid.

[0100] (5) The secondary seed liquid of the above-mentioned Bacillus multi-strain mixture was heated to 10 7 cfu / g (g refers to per gram of Chinese medicine fermentation medium) was added to the Chinese medicine fermentation medium, the water content was adjusted to 50% of the total fermentation system weight, and the fermentation was allowed to stand at 32°C for 5 days.

[0101] Then the first-level seed solution of the Enterococcus faecium strain RY2 was diluted to 10 7 The added amount of cfu / g (per g refers to per gram of traditional Chinese medicine fermentation medium) was added to the above-mentioned traditional Chinese medicine fermentation medium containing Bacillus inaquosorum strain L-14 and Bacillus tequilensis strain 6-9, mixed evenly, and then allowed to stand at 32°C for adsorption for 2 days and then air-dried to obtain a probiotic fermented traditional Chinese medicine preparation.

[0102] 2. Preparation of probiotic fermented Chinese medicinal feed and probiotic-free Chinese medicinal feed for fishery

[0103] Probiotic fermented Chinese medicine feed for fishery: the probiotic fermented Chinese medicine prepared in step 1 (5) above is mixed evenly with the commercially available Tongwei 126 granular grass carp feed, and the probiotic fermented Chinese medicine for fishery is added to the mixture at 1% of the total weight of the feed.

[0104] Chinese herbal medicine feed without probiotics: the mixed Chinese herbal medicine powder prepared in step 1 (1) above is mixed evenly with the commercially available Tongwei 126 granular grass carp feed, and the mixed Chinese herbal medicine powder is added at 1% of the total weight of the feed.

[0105] Example 3: Application of probiotic fermented Chinese medicinal feed to prevent and control bacterial hemorrhagic disease of grass carp caused by Aeromonas hydrophila

[0106] (1) Preparation 10 8 Aeromonas hydrophila bacterial suspension: Aeromonas hydrophila was cultured in LB liquid medium at 37°C and 200 rpm overnight, centrifuged at 5000 rpm for 10 min, collected, counted using a hemocytometer, resuspended in PBS and diluted to a final concentration of 10 8 cfu / mL.

[0107] (2) Feeding: Healthy grass carp (28±3 g), 16 in each group, were fed with the probiotic fermented Chinese medicine feed prepared in Example 2 or ordinary feed (i.e., commercially available Tongwei 126 granular grass carp feed, used as the subsequent challenge control group) for 1 week, once in the morning and once in the evening, with a feeding amount of 3% of body weight.

[0108] (3) Acute intraperitoneal injection of Aeromonas hydrophila: The healthy grass carp were fed for 1 week and then stopped eating for 1 day. They were anesthetized by immersion in MS222 and injected intraperitoneally with 100 μL of 10 8 cfu / mL of Aeromonas hydrophila culture medium, observe continuously and record the death of grass carp.

[0109] An antibiotic control group was also set up in the experiment. Commercially available florfenicol powder for aquaculture was mixed with bait (commercially available Tongwei 126 granular grass carp feed) at 15 mg / kg and fed to grass carp. Grass carp were fed once in the morning and evening, once with antibiotic-added feed and once with ordinary feed (commercially available Tongwei 126 granular grass carp feed) for 5 consecutive days. Normal feed was fed for the rest of the time for a total of 1 week. The feeding amount was 3% of the body weight, and the poisoning was the same as above.

[0110] The results showed that the death time of grass carp in each group was concentrated within 24-48 hours after the infection. As shown in Table 4, the mortality rate of grass carp infected with poison after feeding with ordinary feed for one week was 75%, while the mortality rate of grass carp infected with poison after feeding with fish feed supplemented with probiotic fermented Chinese medicine for one week was 50%, and the protection rate was 33%.

[0111] Table 4. Death rate of grass carp in each group 7 days after infection

[0112] Grouping Number of experimental fish fry Deaths mortality rate Protection rate (%) Poison attack group 16 12 0.75 1% probiotic fermented Chinese medicine group 16 8 0.50 33.33 Antibiotic group 16 11 0.69 8.33

[0113] Example 4: Application of probiotic fermented Chinese medicinal feed to promote grass carp growth and weight gain

[0114] Feeding, healthy grass carp (28 ± 5g), 14 in every group, measure and record the body weight of each fish of each group, feed respectively the probiotic fermentation Chinese medicine feed prepared by embodiment 2 and without probiotic fermentation Chinese medicine feed 3 weeks, respectively feed once in the morning and evening, and feeding amount is 3% of body weight.Experiment arranges antibiotic control group and does not add CK control group simultaneously.Wherein antibiotic control group is that 15mg / kg commercially available aquatic product florfenicol powder mixes commercially available Tongwei 126 particle grass carp feed and throws something and feeds, and grass carp respectively feeds once sooner or later, and continuous feeding antibiotic adds feed 5 days, once a day, drug withdrawal continues after two days, and all the other time feeds common feed (i.e. commercially available Tongwei 126 particle grass carp feed), feeds 3 weeks in a row, and feeding amount is 3% of body weight.CK control group feeds without the common feed (commercially available Tongwei 126 particle grass carp feed) of other added ingredients 3 weeks, and respectively feeds once in the morning and evening, and feeding amount is 3% of body weight. Weigh, measure and record the body weight of each fish in each group after feeding for 3 weeks, and calculate the weight gain rate.

[0115] As shown in Table 5, feeding probiotic fermented Chinese medicine feed for 3 weeks can increase the weight of grass carp, with an average weight gain of 3.6g / fish and a weight gain rate of 12.6%; the average weight gain of Chinese medicine treatment is 0.48g / fish, and the weight gain rate is 1.72%; the average weight gain of antibiotic treatment is 0.58g / fish, and the weight gain rate is 2.707%; the average weight gain of CK treatment is 2.64g / fish, and the weight gain rate is 9.41%.

[0116] Table 5. Effects of probiotic fermented Chinese medicinal feed on growth and weight gain of grass carp

[0117]

[0118] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of a probiotic fermented Chinese medicine preparation for fishery use in the preparation of a product for preventing bacterial hemorrhagic disease in fish, characterized in that: The Chinese medicinal ingredients in the probiotic fermentation Chinese medicinal preparation for fishery use are honeysuckle, dandelion and liquorice; The probiotics in the probiotic fermentation Chinese medicine preparation are Enterococcus faecium ( Enterococcus faecium ) strain RY2, Bacillus tequila ( Bacillus tequilensis ) strains 6-9 and Bacillus aquaticus ( Bacillus inaquosorum ) strain L-14; The Enterococcus faecium ( Enterococcus faecium ) The strain RY2 is registered at the Agricultural Microbiology Center of the China Culture Collection Administration as ACCC 62687; The Bacillus tequila ( Bacillus tequilensis ) strain 6-9 is numbered ACCC 62688 at the Agricultural Microbiology Center of the China Culture Collection Administration; The barren water Bacillus ( Bacillus inaquosorum ) The strain L-14 is numbered ACCC 62689 at the Agricultural Microbiology Center of the China Culture Collection Administration.

2. The use according to claim 1, characterized in that: The probiotic fermented Chinese medicine preparation for fishery also contains fermentation auxiliary materials, which contain corn flour, bran and soybean meal.

3. The use according to claim 2, characterized in that: The probiotic fermented Chinese medicine preparation for fishery use is obtained by inoculating the probiotic into a Chinese medicine fermentation medium for fermentation and culturing; the Chinese medicine fermentation medium contains the Chinese medicine components and the fermentation auxiliary materials.

4. The use according to claim 3, characterized in that: In the traditional Chinese medicine fermentation medium, the weight ratio of honeysuckle, dandelion and liquorice as the traditional Chinese medicine ingredients is 4:2:1; and / or In the traditional Chinese medicine fermentation medium, the weight ratio of corn flour, bran and soybean meal as the fermentation auxiliary materials is 10:4:1; and / or In the traditional Chinese medicine fermentation medium, the weight ratio of the traditional Chinese medicine component to the fermentation auxiliary material is 3:7; and / or The probiotics are inoculated into the traditional Chinese medicine fermentation medium as follows: firstly, the water-poor Bacillus ( Bacillus inaquosorum ) strain L-14 and the Tequila Bacillus ( Bacillus tequilensis ) strains 6-9 were mixed and inoculated into the Chinese medicine fermentation medium, and then inoculated with the Enterococcus faecium ( Enterococcus faecium ) strain RY2; and / or The water-poor Bacillus ( Bacillus inaquosorum ) strain L-14, the Tequila Bacillus ( Bacillus tequilensis ) strains 6-9 and the Enterococcus faecium ( Enterococcus faecium ) The ratio of viable bacteria count of strain RY2 is 1:1:

2.

5. The use according to any one of claims 1 to 4, characterized in that: The probiotic fermentation Chinese medicine preparation for fishery use is prepared according to a method comprising the following steps: (A1) drying and grinding honeysuckle, dandelion, and liquorice, and then mixing them in a weight ratio of 4:2:1 to obtain a mixed Chinese medicine powder; (A2) mixing the mixed Chinese medicinal powder in (A1) with a fermentation auxiliary material in a weight ratio of 3:7 to obtain a Chinese medicinal fermentation medium; the fermentation auxiliary material is a mixture of corn flour, bran, and soybean meal in a weight ratio of 10:4:1; (A3) Set the concentration to 10 8 cfu / mL of the Bacillus subtilis ( Bacillus inaquosorum ) the fermentation broth of strain L-14 and the Tequila Bacillus ( Bacillus tequilensis ) mixing the fermentation culture broths of strains 6-9 at a volume ratio of 1:1 to obtain a Bacillus mixed culture; (A4) The mixed bacterial solution of Bacillus spp. in (A3) was added to a total bacterial count of 10 7 cfu / g was added to the Chinese medicine fermentation medium of (A2), the water content was adjusted to 50%, and the culture was allowed to ferment at 32°C for 3-5 days to obtain the Bacillus Chinese medicine fermentation culture; then the bacterial count was adjusted to 10 7 cfu / g was added to the Bacillus traditional Chinese medicine fermentation culture at a concentration of 10 8 cfu / mL of the Enterococcus faecium ( Enterococcus faecium ) The fermentation culture of strain RY2 was stirred evenly, allowed to stand at 32° C. for 2 days for adsorption, and then air-dried to obtain the probiotic fermentation Chinese medicine preparation for fishery use.

6. The use according to any one of claims 1 to 4, characterized in that: The fish bacterial hemorrhagic disease is caused by Aeromonas hydrophila ( Aeromonas hydrophila ) caused by bacterial hemorrhagic disease in fish.

7. Use of a complete set of products for preparing a probiotic fermented Chinese medicine preparation for fishery use in preparing a product for preventing bacterial hemorrhagic disease in fish, the complete set of products comprising: Honeysuckle, dandelion, liquorice, corn flour, bran, soybean meal, Enterococcus faecium ( Enterococcus faecium ) strain RY2, the Bacillus tequila described in claim 1 ( Bacillus tequilensis ) strains 6-9 and the water-depleting Bacillus described in claim 1 ( Bacillus inaquosorum ) strain L-14.

8. Use of a complete set of products for preparing a probiotic fermented Chinese medicine preparation for fishery use in preparing a product for preventing bacterial hemorrhagic disease in fish, the complete set comprising: The Chinese medicine fermentation medium according to claim 4 or 5, the Enterococcus faecium according to claim 1 ( Enterococcus faecium ) strain RY2, the Bacillus tequila described in claim 1 ( Bacillus tequilensis ) strains 6-9 and the water-depleting Bacillus described in claim 1 ( Bacillus inaquosorum ) strain L-14.

9. The use according to claim 7 or 8, characterized in that: The fish bacterial hemorrhagic disease is caused by Aeromonas hydrophila ( Aeromonas hydrophila ) caused by bacterial hemorrhagic disease in fish.

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