CN116784437A - A kind of fishery probiotic fermented traditional Chinese medicine and its preparation and application - Google Patents

Abstract

The invention discloses a probiotic fermented traditional Chinese medicine for fishery and its preparation and application. A fishery probiotic fermented Chinese medicine preparation, characterized in that: the Chinese medicine ingredients in the fishery probiotic fermented Chinese medicine preparation are honeysuckle, dandelion and licorice; the probiotics in the probiotic fermented Chinese medicine preparation are Hydrobacillus barrens (Bacillus inaquosorum) strain L-14, Bacillus tequilensis strain 6-9 and Enterococcus faecium strain RY2. The probiotic fermented Chinese medicine provided by the invention is added to the feed, which can not only prevent and control fish bacterial hemorrhagic disease, but also promote fish growth.

Description

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

Technical field

The invention relates to the field of microbial fermentation feed additives, and specifically relates to a probiotic fermented traditional Chinese medicine for fishery and its preparation and application.

Background technique

my country is the world's largest producer and consumer of freshwater fish, and the sustainable development of freshwater fisheries is of vital importance. Because the water environment is single, the fish population is large, the disease is highly contagious, and the extensive use of antibiotics has a wide range of harm, so prevention is more important than treatment.

Traditional Chinese medicine can play a role in treating diseases in fishery production due to its natural ingredients, safe efficacy and numerous functions. Probiotics can regulate intestinal function, maintain the ecological balance of intestinal flora, enhance digestion and It has functions such as immunity and growth promotion and is an important component of feed additives. The preparation of fish feed additives by fermenting Chinese medicine with probiotics combines the advantages of both. It uses natural Chinese medicine as the matrix and adds a variety of probiotics for fermentation. On the one hand, it gives full play to the effective active ingredients of traditional Chinese medicine, and on the other hand, it produces a variety of enzymes and Antibacterial secondary metabolites contribute to the digestion, absorption and metabolism of nutrients in fish feed. They can not only effectively prevent and control the occurrence of diseases, but also promote fish growth and weight gain, improve feed conversion rate, and maintain fish intestinal tracts. The homeostasis of microbiota plays a key role in the fishery transformation of reducing resistance and replacing resistance.

Contents of the invention

The object of the present invention is to provide a probiotic fermented traditional Chinese medicine for fishery and its preparation and application.

In the first aspect, the present invention claims a fermented traditional Chinese medicine preparation of fishery probiotics.

The traditional Chinese medicine ingredients in the fishery probiotic fermentation traditional Chinese medicine preparation 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.

The number of the Enterococcus faecium strain RY2 in the Agricultural Microbiology Center of the China Microbial Culture Collection Committee is ACCC 62687;

The number of Bacillus tequilensis strains 6-9 in the Agricultural Microbiology Center of China Microbial Culture Collection Committee is ACCC 62688;

The number of the Bacillus inaquosorum strain L-14 in the Agricultural Microbiology Center of China Microbial Culture Collection Committee is ACCC 62689.

Honeysuckle is the dried flower bud or first-blooming flower of Lonicera japonica Thunb. Sweet and cold in nature. Returns to the lung and stomach meridians. It is mainly used to treat exogenous wind-heat or febrile fever, heat stroke, dysentery due to heat poisoning, carbuncle and boils, laryngeal paralysis, and various infectious diseases.

Dandelion, also known as Huanghuadiding, Bobaoding, Huahualang, etc., is the dried whole plant of the Asteraceae plant Taraxacum mongolicum Hand.-Mazz., Alkaline dandelion (Taraxacum borealisinense Kitag.) or several species of the same genus. Bitter, sweet, cold. Returns to the liver and stomach meridians. Mainly used for boils, mastitis, scrofula, red eyes, sore throat, lung abscess, intestinal abscess, damp-heat jaundice, hot stranguria and astringent pain, etc.

Licorice is the dried root and rhizome of the leguminous plant Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L.. Sweet in taste and neutral in nature. Guixin, lung, spleen, stomach meridians. It has the effects of nourishing the spleen and replenishing qi, clearing heat and detoxifying, eliminating phlegm and relieving cough, relieving pain and relieving pain, and harmonizing various medicines. It is often used to treat weak spleen and stomach, fatigue and fatigue, palpitation and shortness of breath, cough with excessive phlegm, acute pain in the epigastrium and limbs, carbuncle and sore, and relieve the toxicity and potency of drugs.

Furthermore, the fishery probiotic fermented traditional Chinese medicine preparation may also contain fermentation auxiliary materials, and the fermentation auxiliary materials contain corn flour, bran and soybean meal.

Further, the fishery probiotic fermented Chinese medicine preparation can be obtained by inoculating the probiotic bacteria into a Chinese medicine fermentation medium for fermentation and culture; the Chinese medicine fermentation medium contains the Chinese medicine ingredients and the fermentation auxiliary materials.

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

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;

In the traditional Chinese medicine fermentation medium, the weight ratio of the traditional Chinese medicine ingredients and the fermentation auxiliary materials may be 3:7.

Wherein, inoculating the probiotics into the traditional Chinese medicine fermentation medium may be: first inoculating the Bacillus inaquosorum strain L-14 and the Bacillus tequilensis strain 6-9 Mix and inoculate the traditional Chinese medicine fermentation medium, and then inoculate the Enterococcus faecium strain RY2 after culturing.

Further, the Bacillus inaquosorum strain L-14, the Bacillus tequilensis strain 6-9 and the Enterococcus faecium ( Enterococcus faecium) strain RY2 has a viable bacterial count ratio of 1:1:2.

Furthermore, the Bacillus inaquosorum strain L-14, the Bacillus tequilensis strain 6-9 and the Bacillus tequilensis strain 6-9 inserted into each gram of the traditional Chinese medicine fermentation medium The bacterial counts of Enterococcus faecium strain RY2 were 5×10 ⁶ cfu, 5×10 ⁶ cfu and 1×10 ⁷ cfu respectively.

In a specific embodiment of the present invention, the fishery probiotic fermented traditional Chinese medicine preparation can be prepared according to a method including the following steps:

(A1) After drying (such as drying at 60°C) and pulverizing honeysuckle, dandelion and licorice, mix them according to a weight ratio of 4:2:1 to obtain mixed Chinese medicine powder.

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

(A2) Mix the mixed Chinese medicine powder in (A1) and fermentation auxiliary materials in a weight ratio of 3:7 to obtain a Chinese medicine fermentation medium; the fermentation auxiliary materials are made of corn flour, bran and soybean meal in a weight ratio of 10:4:1 weight ratio.

In this step, each component of the traditional Chinese medicine fermentation medium is added into an Erlenmeyer flask according to the proportion, sterilized at 121°C for 30 minutes to effectively kill harmful bacteria in the traditional Chinese medicine fermentation medium, and then cooled to 32°C.

(A3) Combine the fermentation culture liquid (first-level seed liquid) of the Bacillus inaquosorum strain L-14 and the Bacillus tequilensis strain at a concentration of 10 ⁸ cfu/mL 6-9 fermentation culture liquid (primary seed liquid) is mixed according to a volume ratio of 1:1 to obtain a Bacillus mixed bacterial liquid (secondary seed).

(A4) Add the Bacillus mixed bacterial liquid of (A3) to (A2) in an amount such that the total bacterial amount is 1×10 ⁷ cfu/g (each g refers to each gram of the traditional Chinese medicine fermentation medium) The traditional Chinese medicine fermentation culture medium, adjust the water content to 50% (weight percentage), and let it ferment statically at 32° C. for 3-5 days (such as 5 days) to obtain a Bacillus traditional Chinese medicine fermentation culture; and then according to the bacterial count of 1 ^The addition amount ^of faecium) strain RY2, stir evenly, let it sit for adsorption at 32°C for 2 days, and then air-dry to obtain the fishery probiotic fermented traditional Chinese medicine preparation.

In steps (A3) and (A4), the first-level seed liquid of each strain is obtained by inoculating the corresponding strain into the corresponding culture medium and performing single strain enrichment culture to 10 ⁸ cfu/mL.

The three bacterial culture media are as follows:

Bacillus inaquosorum strain L-14, Bacillus tequilensis strain 6-9 and Enterococcus faecium strain RY2 were inoculated into the fermentation liquid medium. The medium formula was as follows: glucose 10g/L, peptone 10g/L, yeast extract 5g/L, KH ₂ PO ₄ 2g/L, MgSO ₄ 2g/L, NaCl 0.5g/L, autoclave at 121°C for 15 minutes. The inoculation amount was inoculated according to the volume ratio of 1:100, and cultured at 37°C for 18-24 hours to obtain a first-level seed liquid with a bacterial concentration of 10 ⁸ cfu/mL.

In a second aspect, the present invention claims a method for preparing fishery probiotic fermented traditional Chinese medicine preparations.

The method for preparing a fishery probiotic fermented traditional Chinese medicine preparation claimed by the present invention may include the steps (A1) to (A4) described above.

In the third aspect, the present invention claims fish feed containing the fishery probiotic fermented traditional Chinese medicine preparation described in the first aspect.

Further, the fish feed of the fishery probiotics fermented Chinese medicine preparation is obtained by adding 1% of the total weight of the fishery probiotics fermented Chinese medicine preparation to the fish feed.

In the fourth aspect, the present invention claims to protect any of the following products or applications:

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

(B2) A complete set of products for preparing probiotic fermented traditional Chinese medicine preparations for fishery use, including: the traditional Chinese medicine fermentation medium described in the first aspect, and the Bacillus inaquosorum strain L described in the first aspect -14. Bacillus tequilensis strain 6-9 described in the first aspect above and Enterococcus faecium strain RY2 described in the first aspect above;

(B3) The fishery probiotic fermented traditional Chinese medicine preparation described in the first aspect above or the fish feed described in the third aspect above or the complete set of products described in (B1) or (B2) are used to prevent fish bacteria. Application in products for sexual hemorrhagic diseases;

(B4) The fishery probiotic fermented traditional Chinese medicine preparation described in the first aspect above or the fish feed described in the third aspect above or the complete set of products described in (B1) or (B2) are used to promote fish growth. applications in products.

In the above aspects, the fish bacterial hemorrhagic disease is a fish bacterial hemorrhagic disease caused by Aeromonas hydrophila (Aeromonashydrophila).

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

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

In a specific embodiment of the present invention, the promotion of fish growth is embodied in the promotion of fish weight gain.

In the above aspects, the fish can be freshwater fish.

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

The technical solution provided by the invention has the following advantages:

(1) The probiotic fermented traditional Chinese medicine feed provided by the present invention uses probiotic fermented traditional Chinese medicine and fishery commonly used feed as raw materials, which fully guarantees the safety of the feed raw materials. The selected traditional Chinese medicine has heat-clearing, detoxifying, and antibacterial tonic activities, and the two are integrated It plays the role of regulating immune balance, antibacterial and anti-inflammatory, and promoting growth and weight gain.

(2) In the probiotic fermented Chinese medicine feed provided by the present invention, the Chinese medicine is dried and crushed, and then fermented with the enriched mixed probiotics, which shortens the fermentation cycle of the Chinese medicine and fully stimulates the active ingredients of the Chinese medicine.

(3) The probiotic fermented traditional Chinese medicine feed provided by the present invention can improve the utilization rate of the feed, increase the feed intake, and promote the animal's nutrient absorption. Digestive enzymes such as protease and cellulase produced by probiotics help break down protein and carbohydrate macromolecules in feed, reduce the digestive burden of farmed animals, and improve feed conversion rate.

(4) The probiotic fermented traditional Chinese medicine feed provided by the present invention can regulate the balance of fish intestinal flora and improve the immunity and disease resistance of animals. Bacillus can produce antibacterial and bacteriostatic active substances, effectively inhibiting the growth of Aeromonas hydrophila, thereby reducing the harm of bacterial pathogen infection; while Enterococcus faecium is adapted to the living environment of the fish intestinal tract, and its large number The 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. It can not only prevent and control grass carp bacterial hemorrhagic disease caused by Aeromonas hydrophila, but also significantly reduce the mortality of grass carp (50%) and increase the protection rate to 33 %, reduce the surface and abdominal symptoms of grass carp, and also promote the growth of grass carp, with an average weight gain of 12.6%.

Description of the drawings

Figure 1 is an Aeromonas hydrophila plate of Bacillus tequilensis strain 6-9 (A) and Bacillus inaquosorum strain L-14 (B) of the present invention. Antagonism diagram.

Figure 2 is a colony morphology diagram of Bacillus tequilensis strain 6-9 (A) and Bacillus inaquosorum strain L-14 (B) of the present invention.

Figure 3 is a phylogenetic tree constructed from the 16S rRNA gene sequences of strains 6-9 and L-14 of the present invention and the model strain of the genus Bacillus.

Figure 4 shows the growth of Bacillus tequilensis strain 6-9 (A) and Bacillus inaquosorum strain L-14 (B) of the present invention under 30°C and different pH conditions for 48 hours. Condition.

Figure 5 shows the bile salt tolerance of Bacillus tequilensis strain 6-9 (A) and Bacillus inaquosorum strain L-14 (B) of the present invention under the conditions of 30°C and pH 7.0. receptive nature.

Figure 6 shows the protease 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. .

Figure 7 shows the fermentation broth of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14 of the present invention cultured for 48 hours at 30°C and pH 7.0 with CMC-Na Cellulase enzyme activity as substrate.

Figure 8 shows the fermentation broth of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14 of the present invention cultured for 48 hours under the conditions of 30°C and pH 7.0, with filter paper as the bottom The cellulase enzyme activity of the substance.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given 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 those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

The Enterococcus faecium strain RY2 in the following examples has been collected in the Agricultural Microbiology Center of the China Microbial Culture Collection Committee, also known as the Chinese Agricultural Culture Collection of Microorganisms, before the filing date of this application. China, referred to as ACCC, address: Institute of Agricultural Resources and Agricultural Zoning, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Haidian District, Beijing, Postcode 100081), strain number is ACCC 62687, collection date is May 17, 2022, self-collection From that date, the public can obtain the strain from the Agricultural Microbiology Center of the China Microbial Culture Collection Committee. ACCC has a special website, the website address is: 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.

The Bacillus tequilensis strains 6-9 in the following examples have been collected in the Agricultural Microbiology Center of the China Microbial Culture Collection and Management Committee, also known as the China Agricultural Microbial Culture Collection and Management Center, before the filing date of this application. Agricultural Culture Collection of China, referred to as ACCC, address: Institute of Agricultural Resources and Agricultural Zoning, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Haidian District, Beijing, Postcode 100081), strain number is ACCC 62688, collection date is May 17, 2022 From the date of collection, the public can obtain the strain from the Agricultural Microbiology Center of the China Microbial Culture Collection Committee. ACCC has a special website, the website address is: 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.

The Bacillus inaquosorum strain L-14 in the following examples has been collected in the Agricultural Microbiology Center of the China Microbial Culture Collection and Management Committee, also known as the China Agricultural Microbial Culture Collection and Management Center (Agricultural), before the filing date of this application. Culture Collection of China, referred to as ACCC, address: Institute of Agricultural Resources and Agricultural Zoning, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Haidian District, Beijing, Postcode 100081), strain number is ACCC 62689, collection date is May 17, 2022 , from the date of collection, the public can obtain this strain from the Agricultural Microbiology Center of the China Microbial Culture Collection Committee. ACCC has a special website, the website address is: 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.

Example 1. Isolation and identification of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14

1. Isolation of bacterial strains

Healthy grass carp (freshwater fish) purchased from fish ponds in Changping District, Beijing and grouper (marine fish) from the seafood market in Ledong City, Hainan Province were anesthetized with MS-222 and then the abdominal wall muscles were cut along the abdominal midline from the anus and cut out. Remove the lateral wall muscles, expose the abdominal cavity, flush the abdominal cavity with sterile PBS buffer, cut the intestines and put them into a 12mL sterile shake tube, add 5mL PBS buffer, fully cut the intestines with sterile surgical scissors, and store at 37°C. Shake at 200r/min for 30min. After the mixture is left to stand for 2 minutes, take 1 mL of the supernatant and make a 10-fold gradient dilution to 10 ^-2 -10 ^-4 . Select three dilution gradients and take 20 μL of the dilution and spread it evenly on the LB solid medium. Each dilution Make three parallel degrees. Place in a 37°C incubator and incubate upside down for 24-48 hours. Colonies of different shapes were picked, and 50 and 23 strains were obtained respectively from the intestines of healthy grass carp and grouper. After three plate passages, stable genetic monoclonal strains were obtained and stored at -80°C.

2. Screening of antagonistic bacteria

1. Pre-screening by plate antagonism method

Aeromonas hydrophila was cultured in LB liquid medium at 37°C and 200 r/min for 24 hours, and then diluted to 10 ⁷ cfu/mL with sterile PBS. Take 20 μL of the diluted bacterial solution and spread it evenly on the LB solid medium. Pick a single colony of the above-mentioned pure bacteria to be tested and draw two short horizontal lines on the plate. Draw three groups on one plate and make two parallel groups. After culturing for 18-24 hours in a constant temperature incubator at 37°C, 37/50 strains of bacteria isolated from the intestines of grass carp and 7/23 strains of bacteria isolated from the intestines of grouper produced obvious inhibition zones.

2. Multiple screening by Oxford cup method

Take a sterile petri dish with a diameter of 9cm, use sterilized 1.5% agar as the base, use tweezers to place 4 sterile Oxford cups on the solidified agar, cool the sterilized LB solid culture medium to 50°C and add The Aeromonas hydrophila culture solution was brought to a final concentration of 10 ⁵ cfu/mL, and then poured into a petri dish with an Oxford cup placed. After the culture medium has solidified, take out the Oxford cup and add 100 μL of the bacterial liquid of the strain to be tested (10 ⁷ cfu/mL) with an inhibitory zone initially screened, using 50 μg/mL chloramphenicol as a control. Place the culture dish in a refrigerator at 4°C for 1 hour to allow the active substances to fully diffuse in the medium, and then incubate the dish 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. As shown in Figure 1, compared with the inhibition zone of chloramphenicol of 17.4±0.5mm, the diameters of the inhibition zones of strain numbers 6-9 and L-14 are 5.3±0.5mm and 9.3±0.2mm, respectively. 30.5% and 53.4% of the antibiotic inhibition zone diameter.

3. Morphological observation and 16S rRNA identification of strains 6-9 and L-14

The strains 6-9 and L-14 stored at -80°C were cultured on LB solid medium at 37°C for 2 days. Observing the morphology of the colonies, the colonies were white, rough and irregular, with bulges and wrinkles, as shown in Figure 2.

The genomic DNA of strains 6-9 and L-14 were extracted respectively, using the genomic DNA of strains 6-9 and L-14 as templates, and using bacterial 16S rRNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5' -GGTTACCTTGTTACGACTT-3') for PCR amplification. The PCR reaction system is shown in Table 1, and the PCR amplification procedure is shown in Table 2. The purity and size of the PCR amplification product were detected by 1.2% agarose gel electrophoresis, and the PCR product with the correct amplification length was sent to Shanghai Sangon Company for sequencing.

Table 1. PCR reaction system

Reagents Dosage (μL) 2×Taqmix 12.5 Primer 27F (10pmol/μL) 1.0 Primer 1492R (10pmol/μL) 1.0 DNA template 0.5 ddH ₂ 0 10.0 total capacity 25.0

Table 2. PCR amplification procedure

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 16S rRNA gene sequence similarity between strain 6-9 and Bacillus tequilensis model strain KJ847721 is 99.66%; the 16S sequence similarity between strain L-14 and Bacillus inaquosorum model strain KCTC13429 is 99.66%. The similarity of rRNA gene sequences was 99.86% respectively. MEGA As shown in Figure 3, the constructed phylogenetic tree shows that strain 6-9 is listed in the same branch as the Bacillus tequilensis model strain KJ847721, and L-14 is listed with the Bacillus inaquosorum model strain KCTC13429. on the same branch.

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

4. Acid-base and bile salt tolerance of strains 6-9 and L-14

Pick single colonies of strains 6-9 and L-14 respectively in 1 mL LB liquid medium, culture in a shaking tube at 37°C and 200 r/min for 24-36 hours, centrifuge at 5000 r/min for 5 min, discard the supernatant, and add sterile PBS. Suspend, measure the OD ₆₀₀ value with a microplate reader, and then adjust the initial concentration of the bacterial solution to an OD ₆₀₀ value of 0.15±0.01.

First add 190 μL of LB liquid culture medium with different pH (3.0, 5.0, 6.0, 7.0, 8.0, 9.0) into the sterile 96-well bacterial culture plate, then add 10 μL of L-14 or 6-9 bacterial liquid, and culture at 37°C for 48 hours. , use a microplate reader to measure the OD ₆₀₀ value, with pH 7.0 as the control, and each treatment was repeated three times. The results are shown in Figure 4. Both strains 6-9 and L-14 grew well under pH 3.0-9.0 conditions, and the optimal growth was pH 5.0-8.0 and pH 6.0-8.0 respectively.

First add 190 μL of LB liquid containing different concentrations (0.03%, 0.05%, 0.1%, 0.2%, 0.3%, % means g/100mL) of porcine bile salt (Solebao G-8310) to the sterile 96-well bacterial culture plate. culture medium, then add 10 μL of strain 6-9 or L-14 bacterial liquid, incubate at 37°C for 48 hours, measure the OD ₆₀₀ value with a microplate reader, and use no bile salt as a control. Each treatment is repeated three times. The results are shown in Figure 5. Strains 6-9 and L-14 have certain bile salt tolerance and can grow at bile salt concentrations of 0.03% and 0.05%.

5. Protease and cellulase activities of fermentation broth of strains 6-9 and L-14

Fermentation medium: glucose 10g/L, peptone 10g/L, yeast extract 5g/L, KH ₂ PO ₄ 2g/L, MgSO ₄ 2g/L, NaCl 0.5g/L, distilled water to 1L, 121°C, 15min Autoclave ready for use.

Strains 6-9 and L-14 were cultured in LB liquid medium in a shaking tube at 37°C and 200r/min for 24 hours, and then inoculated into the fermentation medium with an inoculum volume of 2% (volume percentage) at 37°C and 200r/min. Cultivate for 48 hours, centrifuge at 8000r/min for 15min at 4°C, and take the supernatant as crude enzyme solution.

Prepare reagents and ferment strains L-14 and 6-9 in accordance with the national standard SB/T 10317-1999 "Professional Standard of the People's Republic of China (Protease Activity Assay)" and QB2583-2003 National Standard of the People's Republic of China "Cellulase Preparations" The protease and cellulase activities in the solution 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 6 shows the protease enzyme 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. . Figure 7 shows the fermentation broth of Bacillus tequilensis strain 6-9 and Bacillus inaquosorum strain L-14 of the present invention cultured for 48 hours at 30°C and pH 7.0 with CMC-Na Cellulase enzyme activity as substrate. Figure 8 shows 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 filter paper as the base. cellulase activity of the substance.

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

Example 2. Preparation of fishery probiotic fermented traditional Chinese medicine feed

1. Preparation of probiotic fermented traditional Chinese medicine for fishery use

(1) Preparation of mixed Chinese medicine powder: Dry honeysuckle, dandelion, and licorice at 60°C respectively, crush, and pass through a 40-mesh sieve. Weigh each component in parts by weight, stir evenly, and obtain mixed Chinese medicine powder for later use; each Chinese medicine The ingredients by weight are: 4 parts of honeysuckle, 2 parts of dandelion, and 1 part of licorice.

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

Add each component of the traditional Chinese medicine fermentation medium into an Erlenmeyer flask according to the proportion, sterilize at 121°C for 30 minutes to effectively kill harmful bacteria in the traditional Chinese medicine fermentation medium, and then cool to 32°C.

(3) Prepare a first-level seed liquid of a single strain of probiotics, including Bacillus inaquosorum strain L-14, Bacillus tequilensis strain 6-9 and Enterococcus faecium strain RY2 Inoculate them into the fermentation liquid culture medium respectively, and cultivate the first-level seed liquid of single strains of various probiotics.

The fermentation liquid medium formula is as follows: glucose 10g/L, peptone 10g/L, yeast extract 5g/L, KH ₂ PO ₄ 2g/L, MgSO ₄ 2g/L, NaCl 0.5g/L, sterilized by high pressure at 121°C for 15 minutes. .

The inoculation amount was inoculated according to the volume ratio of 1:100, and cultured at 37°C for 16-24 hours to obtain a first-level seed liquid with a bacterial concentration of 10 ⁷ cfu/mL.

(4) Prepare a second-level seed liquid mixed with multiple strains of Bacillus, and combine the single-strain first-level seeds of the above-mentioned Bacillus inaquosorum strain L-14 and Bacillus tequilensis strain 6-9 liquid (10 ⁷ cfu/mL) at a volume ratio of 1:1 to obtain a second-level mixed Bacillus multi-strain seed liquid.

(5) Add the secondary seed liquid mixed with the above Bacillus multi-strain to the traditional Chinese medicine fermentation medium at an amount of 10 ⁷ cfu/g (each g refers to each gram of the traditional Chinese medicine fermentation medium), and adjust the water content to the total 50% of the weight of the fermentation system was left to ferment at 32°C for 5 days.

Then, the first-level seed liquid of the above-mentioned Enterococcus faecium strain RY2 was added to the above-mentioned Bacillus inaquosorum in an amount of 10 ⁷ cfu/g (each g refers to each gram of Chinese medicine fermentation medium). Strain L-14 and Bacillus tequilensis (Bacillus tequilensis) strain 6-9 were mixed in the traditional Chinese medicine fermentation culture medium, stirred evenly, left to adsorb at 32°C for 2 days, and then air-dried to obtain a probiotic fermented traditional Chinese medicine preparation.

2. Preparation of fishery probiotic fermented traditional Chinese medicine feed and probiotic-free traditional Chinese medicine feed

Fishery probiotic fermented Chinese medicine feed: Mix the probiotic fermented Chinese medicine prepared in the above step 1 (5) with commercially available Tongwei 126 granular grass carp feed, and add fishery probiotic fermented Chinese medicine according to 1% of the total weight of the feed. .

Chinese medicine feed without probiotics: Mix the mixed Chinese medicine powder prepared in the above step one (1) and the commercial Tongwei 126 granular grass carp feed evenly, and add the mixed Chinese medicine powder according to 1% of the total weight of the feed.

Example 3: Using probiotics to ferment traditional Chinese medicine feed to prevent and control grass carp bacterial hemorrhagic disease caused by Aeromonas hydrophila

(1) Prepare 10 ⁸ cfu/mL Aeromonas hydrophila bacterial liquid: Culture Aeromonas hydrophila in LB liquid medium at 37°C, 200r/min overnight, and centrifuge at 5000r/min for 10 minutes to collect the bacteria. The cells were counted with a hemocytometer, resuspended in PBS and diluted to a final concentration of 10 ⁸ cfu/mL.

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

(3) Acute Aeromonas hydrophila challenge by intraperitoneal injection. The above-mentioned healthy grass carp were fed for 1 week and then stopped for 1 day. They were soaked and anesthetized in MS222 and injected intraperitoneally with 100 μL of 10 ⁸ cfu/mL Aeromonas hydrophila liquid. Continuously observe and record the death of grass carp.

The experiment also set up an antibiotic control group. Commercially available aquatic florfenicol powder was fed with 15 mg/kg mixed bait (commercially available Tongwei 126 grain grass carp feed). Grass carp was fed once in the morning and once in the evening, with one antibiotic-added feed and one ordinary feed ( Commercially available Tongwei 126 grain grass carp feed), fed for 5 consecutive days, and fed normal feed for the rest of the time, for a total of 1 week, the feeding amount was 3% of the body weight, and the challenge was the same as above.

The results showed that the death time of grass carp in each group was concentrated within 24-48 hours after challenge. As shown in Table 4, the mortality rate of grass carp challenged with common feed for one week was 75%, while the fish feed added with probiotic fermented traditional Chinese medicine was fed One week later, the mortality rate of grass carp challenged with poison was 50%, and the protection rate was 33%.

Table 4. Death numbers of grass carp in each group after 7 days of challenge

Group Number of experimental fish fry Number of deaths mortality rate Protection rate % Attack team 16 12 0.75 1% probiotic fermented Chinese medicine group 16 8 0.50 33.33 antibiotic group 16 11 0.69 8.33

Example 4: Using probiotics to ferment traditional Chinese medicine feed to promote growth and weight gain of grass carp

Feed healthy grass carp (28±5g), 14 in each group. Measure and record the weight of each fish in each group. Feed the probiotic fermented Chinese medicine feed prepared in Example 2 and the probiotic-free fermented Chinese medicine feed for 3 weeks respectively. Feed once in the morning and once in the evening, and the feeding amount is 3% of the body weight. The experiment also set up an antibiotic control group and a no-CK control group. The antibiotic control group was 15mg/kg commercially available aquatic florfenicol powder mixed with commercially available Tongwei 126 grain grass carp feed. The grass carp was fed once in the morning and once in the evening. The antibiotic-added feed was continuously fed for 5 days, once a day, and the drug was stopped for two days. Continue after the next day, and feed ordinary feed (that is, commercially available Tongwei 126 grain grass carp feed) for the rest of the time for 3 weeks, with the feeding amount being 3% of the body weight. The CK control group was fed ordinary feed without other additives (commercially available Tongwei 126 grain grass carp feed) for 3 weeks, once in the morning and once in the evening, and the feeding amount was 3% of body weight. Weigh, measure and record the weight of each fish in each group after 3 weeks of feeding, and calculate the weight gain rate.

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/carp, and a weight gain rate of 12.6%; treated with Chinese medicine, the average weight gain is 0.48g/carp, with a weight gain rate of 12.6%. Reaching 1.72%; the average weight gain in the antibiotic treatment was 0.58g/strip, and the weight gain rate reached 2.707%; the average weight gain in the CK treatment was 2.64g/strip, and the weight gain rate reached 9.41%.

Table 5. The growth-promoting and weight-increasing effects of probiotic fermented traditional Chinese medicine feed on grass carp

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on 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 scope disclosed in this application and are made using conventional techniques known in the art.

Claims (10)

1. A traditional Chinese medicine preparation fermented by probiotics for fishing is characterized in that: the traditional Chinese medicine components in the traditional Chinese medicine preparation fermented by the fishing probiotics are honeysuckle, dandelion and liquorice;

the probiotics in the probiotic fermented traditional Chinese medicine preparation are enterococcus faecium (Enterococcus faecium) strain RY2, bacillus tertiarygensis (Bacillus tequilensis) strain 6-9 and Bacillus barren (Bacillus inaquosorum) strain L-14;

the enterococcus faecium (Enterococcus faecium) strain RY2 is ACCC 62687 in China Committee for culture Collection of microorganisms;

the Bacillus tefrasis (Bacillus tequilensis) strain 6-9 is ACCC 62688 in China Committee for culture Collection of microorganisms;

the barren bacillus aquaticus (Bacillus inaquosorum) strain L-14 is ACCC 62689 in China Committee for culture Collection of microorganisms.

2. The fish probiotic fermented traditional Chinese medicine preparation according to claim 1, wherein: the traditional Chinese medicine preparation for fish probiotics fermentation also contains fermentation auxiliary materials, wherein the fermentation auxiliary materials contain corn flour, bran and soybean meal.

3. The fish probiotic fermented traditional Chinese medicine preparation according to claim 1 or 2, characterized in that: the traditional Chinese medicine preparation for fermentation of the fish probiotics is obtained by inoculating the probiotics into a traditional Chinese medicine fermentation culture medium for fermentation culture; the traditional Chinese medicine fermentation medium contains the traditional Chinese medicine components and the fermentation auxiliary materials.

4. The fish probiotic fermented traditional Chinese medicine preparation according to claim 3, wherein: in the traditional Chinese medicine fermentation medium, the weight ratio of honeysuckle, dandelion and liquorice serving as the traditional Chinese medicine components is 4:2:1;

and/or

In the traditional Chinese medicine fermentation medium, the weight ratio of corn flour, bran and soybean meal serving as fermentation auxiliary materials is 10:4:1;

and/or

In the traditional Chinese medicine fermentation medium, the weight ratio of the traditional Chinese medicine components to the fermentation auxiliary materials is 3:7;

and/or

Inoculating the probiotics into the traditional Chinese medicine fermentation medium comprises the following steps: firstly, mixing and inoculating the barren bacillus aquaticus (Bacillus inaquosorum) strain L-14 and the bacillus tertageus (Bacillus tequilensis) strain 6-9 into the traditional Chinese medicine fermentation medium, and inoculating the enterococcus faecium (Enterococcus faecium) strain RY2 after culturing;

further, the viable count ratio of the barren bacillus aquaticus (Bacillus inaquosorum) strain L-14, the bacillus tervelarius (Bacillus tequilensis) strain 6-9 and the enterococcus faecium (Enterococcus faecium) strain RY2 which are inoculated into the traditional Chinese medicine fermentation medium is 1:1:2.

5. The fish probiotic fermented traditional Chinese medicine preparation according to any one of claims 1 to 4, characterized in that: the traditional Chinese medicine preparation for fish probiotics fermentation is prepared by the following steps:

(A1) Drying and crushing honeysuckle, dandelion and liquorice, and mixing according to the weight ratio of 4:2:1 to obtain mixed traditional Chinese medicine powder;

(A2) Mixing the mixed traditional Chinese medicine powder in the step (A1) with fermentation auxiliary materials according to the weight ratio of 3:7 to obtain a traditional Chinese medicine fermentation medium; the fermentation auxiliary material is formed by mixing corn flour, bran and bean pulp according to the weight ratio of 10:4:1;

(A3) The concentrations were all 10 ⁸ cfu/mL of the fermentation culture solution of the barren bacillus aquaticus (Bacillus inaquosorum) strain L-14 and the fermentation culture solution of the bacillus tefraxinus (Bacillus tequilensis) strain 6-9 are mixed according to the volume ratio of 1:1 to obtain a bacillus mixed bacterial solution;

(A4) The bacillus mixed bacterial liquid of (A3) is mixed according to the total bacterial amount of 10 ⁷ Adding cfu/g into the traditional Chinese medicine fermentation culture medium of (A2), adjusting the water content to 50%, and standing and fermenting at 32 ℃ for 3-5 days to obtain a bacillus traditional Chinese medicine fermentation culture; then according to the bacterial load of 10 ⁷ The addition amount of cfu/g is 10 percent of the concentration of the bacillus Chinese medicine fermentation culture ⁸ cfu/mL of fermentation culture solution of the enterococcus faecium (Enterococcus faecium) strain RY2 is stirred uniformly, then is subjected to standing adsorption at 32 ℃ for 2 days, and is then air-dried, so that the traditional Chinese medicine preparation fermented by the probiotics for fishing is obtained.

6. A method for preparing a fish probiotic fermented traditional Chinese medicine preparation, comprising the steps (A1) to (A4) described in claim 5.

7. A fish feed comprising the probiotic fermented traditional Chinese medicine preparation for fishing according to any one of claims 1 to 5.

8. The fish feed of claim 7, wherein: the fish feed of the fish probiotic fermented traditional Chinese medicine preparation is obtained by adding the fish probiotic fermented traditional Chinese medicine preparation with the total weight of 1% to the fish feed.

9. Any one of the following products or applications:

(B1) A kit for preparing a probiotic fermented traditional Chinese medicine preparation for fishing comprising: honeysuckle, dandelion, licorice, corn flour, bran, soybean meal, enterococcus faecium (Enterococcus faecium) strain RY2 as claimed in claim 1, bacillus tertageus (Bacillus tequilensis) strain 6-9 as claimed in claim 1 and Bacillus barrens (Bacillus inaquosorum) strain L-14 as claimed in claim 1;

(B2) A kit for preparing a probiotic fermented traditional Chinese medicine preparation for fishing comprising: the traditional Chinese medicine fermentation medium of claim 4 or 5, the enterococcus faecium (Enterococcus faecium) strain RY2 of claim 1, the bacillus tervelarius (Bacillus tequilensis) strain 6-9 of claim 1, and the bacillus barrens (Bacillus inaquosorum) strain L-14 of claim 1;

(B3) Use of a probiotic fermented traditional Chinese medicine preparation for fish according to any one of claims 1 to 5 or a fish feed according to claim 7 or 8 or a kit of products according to (B1) or (B2) for the preparation of a product for preventing bacterial hemorrhagic disease in fish;

(B4) Use of a probiotic fermented traditional Chinese medicine preparation for fish according to any one of claims 1 to 5 or a fish feed according to claim 7 or 8 or a kit of products according to (B1) or (B2) for the preparation of a product for promoting fish growth.

10. The use according to claim 9, characterized in that: the bacterial hemorrhagic disease of fish is bacterial hemorrhagic disease of fish caused by aeromonas hydrophila (Aeromonas hydrophila).