CN115074281B - Ginseng bacillus capable of producing organic acid at high yield and application of ginseng bacillus in microecological preparation - Google Patents

Abstract

The application discloses a ginseng bacillus strain capable of producing organic acid at high yield and application of the ginseng bacillus strain in a microecological preparation, and belongs to the technical field of microecological preparations. The bacillus ginseng (bacillus gingiensis) BG2204 is preserved in the China center for type culture collection, and the preservation number is CCTCC NO: m2022496, the preservation address is Wuhan, university of Wuhan, and the preservation date is 2022, 04 and 26. The application prepares the ginseng bacillus into fermented feed and microecological preparation through optimizing the fermentation culture and acid production capacity of the ginseng bacillus, verifies the effect in aquaculture and broiler chicken feeding, and provides theoretical basis for developing the ginseng bacillus into microecological preparation to be applied to the fields of livestock breeding, environmental management and the like.

Description

A ginseng Bacillus strain with high organic acid production and its application in microecological preparations

Technical field

The present invention relates to the technical field of microecological preparations, and in particular to a strain of Bacillus ginseng with high organic acid production and its application in microecological preparations.

Background technique

Probiotics (also known as live bacteria preparations, microbial inoculants) are live microbial preparations made from normal microorganisms or substances that promote the growth of microorganisms. At present, microecological preparations have been widely used in various fields such as livestock breeding, agricultural planting, medical care, healthy food, and environmental management.

Bacillus and Lactobacillus are the most commonly used types of microbial preparations. Bacillus has good tolerance and can produce abundant enzymes; Lactobacillus metabolizes to produce lactic acid, which can inhibit the growth and reproduction of harmful bacteria and has good microbial properties. Ecological regulation. In the fields of animal husbandry, health food, etc., they can be used as probiotics to produce fermented foods, feeds and microecological preparations, which can improve feed utilization, increase food flavor, improve intestinal microecological balance, enhance host immunity, and improve host immunity. Growth performance and health levels. In terms of environmental management, Bacillus and Lactobacillus can also decompose organic matter, regulate water quality and microecological balance.

Grains, legumes, and oilseeds contain phytic acid, which binds phosphorus, rendering 70%-80% of it indigestible. This limits the use of these crops in feed, especially in the development of high-concentration diets for intensive livestock and poultry farming. Phytic acid is a special chemical derivative of the hexafold inositol, which has six hydroxyl groups bound to six phosphate residues. Phosphate residues are chemically active and can bind to metal ions (calcium, sodium, potassium, zinc, and copper). Phytic acid may also react with amino acid residues, rendering them unavailable to plants. Thus, in addition to serving as a reservoir for phosphorus, phytic acid binds a large portion of trace elements, proteins, carbohydrates and amino acids, converting them into complex insoluble aggregates. Phytase (myo-inositolexakisporate phosporlase) is a special type of phosphomonoesterase that can hydrolyze phytic acid to release phosphoric acid. In the digestive tract of pigs, poultry, and other monogastric animals, the production of phytase is very limited. Most microbial phytases are intracellular enzymes, but fungi, bacteria of the genus Enterobacter and Bacillus can produce secreted phytase, which has promoted the widespread application of phytase in industry and agriculture.

AnnaA et al. discovered the phytase encoding gene in Bacillus ginseng M2.11 in 2015, revealing that this strain can secrete phytase. However, Bacillus species that can both hydrolyze phytic acid and produce high organic acids, especially Bacillus ginseng, have not been reported.

Contents of the invention

The purpose of the present invention is to provide a strain of Bacillus ginseng with high organic acid production and its application in microecological preparations. By optimizing its fermentation culture and acid production capacity, it can be prepared into fermented feed and microecological preparations and verified. The effect in aquaculture and broiler chicken feeding provides a theoretical basis for the development of microecological preparations for use in livestock breeding, environmental management and other fields.

In order to achieve the above objects, the present invention provides the following solutions:

The present invention provides a strain of Bacillus ginsengihumi BG2204, which has been deposited in the China Typical Culture Collection Center. The deposit number is CCTCC NO: M2022496. The deposit address is Wuhan University, Wuhan, China. The deposit date is April 26, 2022. day.

The invention also provides a method for cultivating Bacillus ginseng with high lactic acid production. The Bacillus ginseng is activated and inoculated into a fermentation medium, cultured at 35°C to 40°C for 18h to 22h, and molasses, corn steep liquor and calcium carbonate are added. Continue culturing at 40°C to 50°C for 20h to 24h.

Further, the components of the fermentation medium include sucrose 20g/L~40g/L, soybean meal 5g/L~10g/L, yeast extract 5g/L~10g/L, and zinc sulfate 0.5g/L~1g/L. L, magnesium sulfate 0.5g/L~1g/L.

The invention also provides a high-density cultivation method of the Bacillus ginseng, which involves activating and culturing the Bacillus ginseng, and inoculating the obtained seed liquid into a fermentation culture liquid for fermentation, and the fermentation culture liquid is produced from the fermentation culture medium. The culture medium and water are mixed at a ratio of 31.75g:1.5L, and then the variable speed feeding method is used to supplement the carbon source and nitrogen source in batches.

Further, when the volume of the fermentation culture liquid is 1.5L, the specific fed-batch method is:

For fermentation of 0h to 2h, no carbon source is added, and the speed is kept above the minimum speed of 100 rpm/min; for fermentation of 2h to 6h, the flow acceleration of the carbon source is 3% to 6% of the total volume of the fed fermentation culture liquid per hour. The rotation speed is controlled at 120rpm/min ~ 200rpm/min; fermentation is 6h ~ 14h, the flow acceleration of the fed carbon source is 5% ~ 10% of the total volume of the fed fermentation culture liquid per hour, and the rotational speed is controlled at 200rpm/min ~ 350rpm/min ; Fermentation 14h ~ 20h, the flow acceleration of the fed carbon source is 5% ~ 10% of the total volume of the fermentation culture liquid per hour, and the rotation speed is controlled at 120rpm/min ~ 200rpm/min; in the final stage of fermentation, 20h ~ 22h, stop the flow Add carbon source;

Fermentation 0h~2h, no compound nitrogen source is added; fermentation 2h~6h, the flow acceleration of the compound nitrogen source is 1%~3% of the total volume of the fermentation culture medium per hour; fermentation 6h~14h, compound nitrogen source The flow acceleration is 3% to 10% of the total volume of the fed fermentation culture liquid per hour; for 14h to 20h fermentation, the flow acceleration of the composite nitrogen source is 1% to 3% of the total volume of the fed fermentation culture liquid per hour. The entire fermentation The dissolved oxygen content during the process is controlled to be above 25% to 45%; in the final stage of fermentation for 20h to 22h, the feeding of compound nitrogen sources is stopped.

Further, the fermentation temperature is 35°C to 40°C, and the fermentation period is 18h to 22h. .

Further, the fed carbon source includes 400g to 500g of sucrose, and the fed volume is 1.8L; the fed compound nitrogen source includes yeast extract powder and soybean meal 100g to 150g with a mass ratio of 1:1, and the fed volume is 1.8L. 3L.

The present invention also provides a bacterial powder obtained according to the high-density culture method. The bacterial powder is obtained by centrifugal drying of the fermented bacterial liquid.

The invention also provides a fermented feed. In parts by weight, the raw materials of the feed include: 60 parts of corn straw with a moisture content of 15%, 1 part of 100,000 U/g xylanase, 20 parts of fermented feed fermentation liquid and 50 parts of bran water;

Wherein, the fermented feed fermentation liquid is prepared by the following method: 120g of molasses, 30g of corn flour, 1L of bran water are mixed, boiled for 30 minutes, then diluted to 1L, sterilized at 121°C for 30 minutes, and then inoculated with 5% of Bacillus ginseng seed liquid , 37°C, 180rpm/min for 22h; the mass concentration of the bran water is 10%;

The fermented feed is prepared by mixing the raw materials, putting them into sealed bags, and anaerobically fermenting them at 37°C for 5 days.

The invention also provides a microecological preparation containing the ginseng Bacillus, including the following components in parts by weight: 2 parts of the fermented feed fermentation liquid, 2 parts of chitosan oligosaccharide, 1 part of trehalose, zeolite Powder 3 parts.

The invention also provides an application of the Bacillus ginseng or the microecological agent in aquaculture and feeding broilers.

The invention discloses the following technical effects:

(1) The present invention separates and screens a strain of Bacillus ginseng with high organic acid production from liquor yellow water. Through the optimization of culture medium and fermentation process, the number of viable bacteria of Bacillus ginseng reaches 3.5×10 ⁹ CFU/mL after optimization.

(2) The present invention adopts a two-step fermentation method and utilizes molasses, corn steep liquor, soybean meal and other raw materials to significantly increase the lactic acid production of Bacillus ginseng, and the lactic acid production reaches 2g/L to 4g/L.

(3) The high-quality fermented feed containing Bacillus ginseng produced by the present invention contains organic acids such as lactic acid and acetic acid. Through the rational utilization of industrial by-products molasses and agricultural waste corn straw, solid waste can be optimally utilized to achieve energy conservation and emission reduction.

(4) The present invention applies Bacillus ginseng to aquaculture and the feeding of broiler chickens, which significantly improves the absorption of feed by crucian carp and improves the immunity of crucian carp; significantly reduces the diarrhea rate and feed-to-meat ratio of broiler chickens, reaching replace antibiotics. It provides a theoretical basis for developing microecological preparations for use in livestock breeding, environmental management and other fields.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exerting creative efforts.

Figure 1 shows the colony morphology of Bacillus ginseng;

Figure 2 is a hydrolysis diagram of Bacillus ginseng calcium phytate;

Figure 3 is a Gram stain diagram of Bacillus ginseng;

Figure 4 is a graph showing the lactic acid content of Bacillus ginseng;

Figure 5 is a screening diagram for the optimization of the types and addition amounts of carbon sources in the fermentation of Bacillus ginseng, where a is the growth of the cells under different types of carbon sources, and b is the growth of the cells under different addition amounts using sucrose as the carbon source;

Figure 6 is a diagram showing the optimization and screening of nitrogen source types in Bacillus ginseng fermentation;

Figure 7 is a screening diagram for optimizing the amount of nitrogen source added in the fermentation of Bacillus ginseng, where a is the growth of the cells under different additions of magnesium sulfate, and b is the growth of the cells under different additions of zinc sulfate;

Figure 8 is a screening diagram for optimizing the types and amounts of inorganic salts used in Bacillus ginseng fermentation;

Figure 9 is a screening diagram for optimizing the fermentation conditions of Bacillus ginseng, where a is the measured value of OD ₆₀₀ in the bacterial liquid during different fermentation cycles, b is the number of viable bacteria in the bacterial liquid under different initial fermentation pH, and c is the bacterial liquid under different fermentation temperatures. The number of viable bacteria, d is the number of viable bacteria in the bacterial solution under different shaking flask rotation speeds, e is the number of viable bacteria in the bacterial solution under different inoculum volumes, and f is the number of viable bacteria in the bacterial solution under different filling volumes.

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range and any other stated value or value intermediate within a stated range is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples are intended to be illustrative only.

The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to.

Unless otherwise specified, the reagents and materials used in the examples are all commonly purchased by those skilled in the art.

Example 1 Screening and identification of Bacillus ginseng

1. Screening of bacterial strains

First, take 0.5 mL of white wine yellow water, select 10 ^-6 dilution according to the dilution coating method, and apply it on the plate. The coating volume is 100 μL. Incubate at 37°C for 24 hours. Select strains with larger diameters that produce yellow transparent circles for plate streaking. After re-screening, a single colony was finally selected and inoculated into YPD liquid medium for enrichment culture at 37°C and 180 rpm/min.

2. Identification of bacterial strains

2.1 Morphological identification

This strain was streaked on a YPD plate. The colonies of this strain were light yellow in the middle and white at the edge. The surface was smooth, moist, round, and the colony diameter was about 2 mm (Figure 1).

Gram staining was performed on the strain: it was observed through an optical microscope and an oil lens that the strain was a Gram-positive bacterium and was sporulated (Figure 3).

2.2 Identification of phytase production ability

The bacterial solution was inoculated on double-layer YPD plates with 0.25g, 0.5g, 0.75g, and 1.0g calcium phytate added. It was observed that the strain has the ability to hydrolyze calcium phytate. The diameters of the transparent circles are 23mm, 20mm, and 19mm, 19mm (Figure 2).

2.3 Identification of organic acid production ability

The supernatant after centrifugation of the bacterial solution is passed through a membrane and then analyzed by high performance liquid chromatography. The chromatographic column is BIO-RAD's Aminex HPX-87H (300mm×7.8mm). This chromatographic column can separate parts of a single sample at one time. Organic acids, according to Figure 4, it can be seen that organic acids such as lactic acid, acetic acid and propionic acid are produced in the bacterial solution. The peak time of lactic acid is 13min, the peak time of acetic acid is 15min, and the peak time of propionic acid is 18min. In Figure 4, 13.406 The peak at 15.701 is lactic acid, the peak at 15.701 is acetic acid, and the peak at 18.524 is propionic acid. According to the lactic acid standard curve y=253019x-5161 (where y is the peak area and x is the lactic acid content), the lactic acid content is calculated to be about 1g/L.

2.4 Molecular biology identification

The DNA of this strain was extracted and amplified using bacterial universal primers, and then 16SrDNA sequencing analysis was performed to obtain a partial sequence of the 16srDNA gene as shown in SEQ ID NO.1.

SEQ ID NO.1：

TGGCGGCGTGCTATAATGCAAGTCGAGCGAACTGATGAAGAGCTTGCTTTTGATCAGGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTAGGATAACTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGCATGGAGAGAGATTGAAAGATGGCTTCGGCTATCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGC GTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGGTAATACGTAGGT GGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGATGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAG TCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCTCCCTAGAGATAGGGCCTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGT CGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAGACCGCGAGGTTAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTG CATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGACAGAGTG.

Through NCBI sequence comparison, the results showed that it has the highest similarity with Bacillus ginseng, reaching 99.9%, and the strain was determined to be Bacillus ginseng. The strain has been sent to the China Type Culture Collection Center for preservation on April 26, 2022. It is classified as Bacillus ginsengihumi BG2204, the preservation number is CCTCC NO: M2022496, and the preservation address is Wuhan University, Wuhan, China.

Example 2 Optimization of culture medium, culture conditions and high-density fermentation of Bacillus ginseng

1. Strains

Bacillus ginseng BG2204 isolated and identified in Example 1.

2. Optimization of culture media and culture conditions

Bacteria activation medium: 20g/L glucose, 20g/L tryptone, 10g/L yeast extract powder; 37°C, 180rpm/min, culture for 24 hours.

2.1 Optimization of carbon source types

Optimize the selection of carbon source types on the above culture medium. Carbon source types include: α-lactose, glucose, sucrose, soluble starch, D-xylose, and β-cyclodextrin. The initial addition amount is selected from 5g/L to 10g/L. ; Record the number of viable bacteria after adding each carbon source. It can be seen from Figure 5 that the number of viable bacteria is the highest after adding sucrose, so the optimal carbon source is sucrose.

Optimize the addition amount of the optimal carbon source, and select 5g/L, 10g/L, 20g/L, 30g/L, and 40g/L respectively for the addition amount optimization experiment; as shown in Figure 5, when the addition amount is 30g/L, The number of viable bacteria is the highest, so the optimal addition amount is 30g/L.

2.2 Optimization of nitrogen source types

Optimize the nitrogen source types on the culture medium finalized in Section 2.1. The nitrogen source types include: tryptone, yeast extract powder, peptone, corn flour, ammonium sulfate, urea, ammonium chloride, sodium nitrate, soybean meal, initial addition amount It is 10g/L; record the number of viable bacteria after adding each nitrogen source. As can be seen from Figure 6, the number of viable bacteria was the highest after adding yeast extract powder, soybean meal, and tryptone. Therefore, the nitrogen sources yeast extract powder, soybean meal, and tryptone were selected for subsequent optimization of the composition and ratio of the composite nitrogen source.

Optimize the composition and ratio of the compound nitrogen source. The selected ratios are yeast extract powder: tryptone = 1:1, 1:2, 2:1, soybean meal: yeast extract powder = 1:1, 1:2, 2:1. Soybean meal: tryptone = 1:1, 1:2, 2:1, record the number of viable bacteria after culture under each addition condition, see Table 1 for details.

Table 1 Optimization of the proportion of composite nitrogen sources

It can be seen from Table 1 that when soybean meal: yeast extract powder = 1:1, the number of viable bacteria is the highest, so soybean meal: yeast extract powder = 1:1 is selected as the composite optimal nitrogen source composition and ratio.

The total addition amount of the above-mentioned optimal composite nitrogen source was optimized, and 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, and 30g/L were selected for the addition amount optimization experiment; as can be seen from Figure 6, When the compound nitrogen source addition amount is 20g/L, the number of viable bacteria is the highest, so 20g/L is selected as the optimal compound nitrogen source addition amount.

2.3 Optimization of inorganic salt types

Optimize the selection of inorganic salt types on the culture medium finalized in Section 2.2. Inorganic salt types include: potassium dihydrogen phosphate, sodium chloride, magnesium sulfate, calcium chloride or zinc sulfate. The initial addition amount is 1.0g/L; record The number of viable bacteria after the addition of each inorganic salt. It can be seen from Figure 7 that when the inorganic salts are magnesium sulfate and zinc sulfate, the number of viable bacteria is higher, so magnesium sulfate and zinc sulfate are selected as the optimal inorganic salts.

To optimize the addition amount of the above-mentioned optimal inorganic salts, magnesium sulfate and zinc sulfate were selected for the addition amount optimization experiment, and 0.25g/L, 0.5g/L, 0.75g/L, 1.0g/L, 1.5g/ L was tested. It can be seen from Figure 8 that when the amount of magnesium sulfate added is 1.0g/L and the amount of zinc sulfate added is 0.75g/L, the number of viable bacteria is higher, so magnesium sulfate 1.0g/L and zinc sulfate 0.75g are selected respectively. /L is the optimal inorganic salt addition amount.

2.4 Optimization of fermentation conditions

The strain fermentation conditions (fermentation time, initial fermentation pH, fermentation temperature, shaker speed, inoculum volume and liquid filling volume) were optimized based on the above-mentioned optimized carbon source, nitrogen source and optimized medium of inorganic salts.

The preferred embodiment of the fermentation cycle is: measure its OD value every 2 hours starting from 0h; the preferred embodiment of the initial fermentation pH is: 3, 4, 5, 6, 7, 8, 9; the preferred embodiment of the temperature is: 25°C , 30°C, 35°C, 40°C, 45°C; the preferred embodiments of the shaking table rotation speed are: 140rpm/min, 160rpm/min, 180rpm/min, 200rpm/min, 220rpm/min; the preferred embodiments of the inoculation amount are: 2%, 4%, 6%, 8%, 10%; the preferred embodiments of the liquid volume are: 25mL/250mL, 50mL/250mL, 75mL/250mL, 100mL/250mL, 125mL/250mL;

It can be seen from Figure 9 that when the fermentation cycle is 18h ~ 22h; the initial fermentation pH is 5 ~ 7; the fermentation temperature is 35°C ~ 40°C; the shaker speed is 160rpm/min ~ 220rpm/min; the inoculum amount is 2% ~ 4% ; When the liquid volume is 30mL/250mL~100mL/250mL, the number of viable bacteria is the highest, reaching 3.5×10 ⁹ CFU/mL.

High-density fermentation of Bacillus ginseng in 3.10L fermentation tank

The components of the secondary seed culture medium are the same as the activation culture medium in Example 2, specifically: 20g/L glucose, 20g/L tryptone, 10g/L yeast extract powder; culture at 37°C, 180rpm/min for 24 hours.

The bottom water of the fermentation tank at the level of the 10L fermentation tank contains: 1.5L of water, 20g of sucrose, 5g of yeast extract, 5g of soybean meal, 0.75g of zinc sulfate, 1.0g of magnesium sulfate, and the pH is adjusted to 5.0 to 7.0.

Preparation of the fed carbon source: The volume of the fed carbon source is 1.8L, the composition is 400g~500g of sucrose, and it is sterilized at 115°C for 20 minutes.

Preparation of fed nitrogen source: The volume of fed nitrogen source is 3L, including 1.5L nitrogen source containing yeast extract powder (100g~150g) and 1.5L nitrogen source containing soybean meal (100g~150g), sterilized at 115°C for 20 minutes .

3.1 Fermentation culture

Multi-stage fed-batch fermentation: Put the Bacillus ginseng seed liquid prepared in the secondary seed culture medium into a 10L fully automatic fermentation tank filled with 1.5L bottom water at 3% (V/V) of the tank volume. The temperature is 35℃～40℃, and the fermentation period is 18h～22h. Fermentation for 0h~2h, no carbon source is added, and the speed is kept above the minimum speed of 100rpm/min; fermentation for 2h~6h, the carbon source flow acceleration is 40mL/h-90mL/h, and the speed is controlled at 120rpm/min~200rpm/min; fermentation 6h～14h, the carbon source flow acceleration is 60mL/h-120mL/h, and the rotation speed is controlled at 200rpm/min～350rpm/min; fermentation 14h～20h, the carbon source flow acceleration is 40mL/h-90mL/h, and the rotation speed is controlled at 120rpm/min ~200rpm/min; in the final stage of fermentation, 20h~22h, stop adding carbon source.

For fermentation 0h~2h, no compound nitrogen source is added; for fermentation 2h~6h, the flow acceleration of the compound nitrogen source is: 20mL/h-50mL/h; for fermentation 6h~14h, the flow acceleration of the compound nitrogen source is: 50mL/h-280mL/h; During the fermentation period of 14h to 20h, the flow acceleration of the compound nitrogen source is: 40mL/h-80mL/h, and the dissolved oxygen content during the entire fermentation process is controlled above 25% to 45%; in the final stage of fermentation, from 20h to 22h, the flow of the compound nitrogen source is stopped.

3.2 Preparation of bacteria powder

Centrifuge the fermented bacteria liquid at 8000rpm/min for 15min~20min, collect 140g~180g of bacteria, and spray-dry the bacteria at an inlet temperature of 110°C~140°C and an outlet temperature of 60°C~70°C to obtain 14g~18g of bacteria powder.

3.3 Results

The final viable bacterial count of the bacterial powder obtained was 3×10 ¹⁰ CFU/g.

Comparative example 1

The implementation method is the same as Example 2, but compared with Example 2, the optimization process only lacks optimization of carbon source types and addition amounts, and the number of viable bacteria is measured after the fermentation is completed. The result was: 1.3×10 ⁹ CFU/mL.

Comparative example 2

The implementation method is the same as Example 2, but compared with Example 2, the optimization process only lacks optimization of nitrogen source types and addition amounts, and the number of viable bacteria is measured after the fermentation is completed. The result was: 1.9×10 ⁹ CFU/mL.

Comparative example 3

The implementation method is the same as Example 2, but compared with Example 2, the optimization process only lacks optimization of the type and addition amount of inorganic salts, and the number of viable bacteria is measured after the fermentation is completed. The result was: 1.1×10 ⁹ CFU/mL.

Example 3 Optimization of acid production ability of Bacillus ginseng

1. Bacterial strain: same as Example 2

2. Culture medium:

(1) The bacterial strain activation liquid culture is carried out according to the conditions optimized in Example 2. After the activation culture, the lactic acid content in the bacterial liquid is measured.

(2) The activated bacterial liquid is moved to the fermentation medium to continue culturing.

The components of the fermentation medium are: sucrose 20g/L~40g/L, soybean meal 5g/L~10g/L, yeast extract 5g/L~10g/L, zinc sulfate 0.5g/L~1g/L, magnesium sulfate 0.5 g/L～1g/L. The culture temperature is 35℃~40℃, culture for 18h~22h, then add 30g/L~50g/L molasses, corn steep liquor 5g/L~10g/L, calcium carbonate 5g/L~10g/L, 40 ℃ ~ 50 ℃, continue to culture for 20h ~ 24h, and then measure the lactic acid content.

The lactic acid content in the above steps was measured using high performance liquid chromatography. The measurement results showed that the lactic acid content (1g/L~2g/L) in the activation medium increased approximately twice to 2g/L~4g/L.

Example 4 Preparation of Bacillus ginseng fermented feed

(1) The bacterial strain activation liquid medium is the same as the bacterial strain activation medium in Example 2;

(2) Preparation of fermented feed fermentation liquid:

120g of molasses, 30g of corn flour, add 1L of bran water (10% bran), add water and boil for 30 minutes, then adjust the volume to 1L, sterilize at 121°C for 30 minutes, and then inoculate 5% of Bacillus ginseng seed liquid (strain activation liquid culture (obtained later), cultured at 37°C, 180rpm/min for 22h. The number of viable bacteria of Bacillus ginseng in the seed liquid was measured to be 3×10 ⁸ CFU/mL;

(3) Fermented feed preparation method:

In parts by weight, 60 parts of corn straw (moisture content 15%), 1 part of xylanase (100,000 U/g), 20 parts of fermented feed fermentation liquid, 50 parts of bran water, mix well and put into a sealed bag , anaerobic fermentation at 37°C for 5 days to obtain Bacillus ginseng fermented feed.

The various indicators in the fermented feed tested after 5 days were: ginseng Bacillus 5.4×10 ⁹ CFU/g, lactic acid content 5.39%, acetic acid content 3.94%, and acidity 9.76%. The increase in acetic acid content in fermented feed can effectively inhibit the growth of mold spores in the feed and effectively prevent feed mildew; and lactic acid can change the intestinal environment in the animal intestines and adjust the balance of gastrointestinal flora.

Example 5 Application of Bacillus ginseng inoculant in aquaculture

Preparation of microecological preparation: In parts by weight, 2 parts of the fermented feed fermentation liquid in Example 4, 2 parts of chitosan oligosaccharide, 1 part of trehalose, and 3 parts of zeolite powder.

Mix the fermented feed fermentation liquid (number of viable bacteria 3×10 ⁸ CFU/mL) with chitosan oligosaccharide, trehalose and lime powder, stir evenly, and pour it into the pond according to different addition amounts. The crucian carp cultured in the experiment was purchased from the wet market of Huazhong Agricultural University in Wuhan.

Before the start of the experiment, fish were purchased and temporarily raised in the pond for 15 days as a pre-feeding period. Then 600 fish were randomly collected from the pond and divided into a control group, a low-dose experimental group and a high-dose experimental group (200 fish in each group, average weight: 0.52±0.18 g, average body length: 3.44±0.37 cm), each group Set up three repetitions. Cultivated in ponds.

The amount of microecological preparations added in the low-dose experimental group was 250g/mu (the number of viable bacterial powders was not less than 3×10 ⁸ CFU/g), and the amount of microecological preparations added in the high-dose experimental group was 500g/mu (the number of viable bacterial powders was not less than 3×10 8 CFU/g). The bacterial count is not less than 3×10 ⁸ CFU/g), and no microbial ecological preparations are added to the control group. During the official period, weight gain and survival numbers were recorded every day. The results are shown in Table 2.

Table 2 Results of feeding crucian carp with Bacillus ginseng bacteria

It can be seen from Table 2 that Bacillus ginseng inoculum can increase the weight gain rate (increased by 336%) and survival rate (increased by 7.14%). This shows that the bacterial agent can not only improve the absorption of feed by crucian carp, but also improve the immunity of crucian carp.

Example 6 Application of Bacillus ginseng inoculant in raising broilers

The Bacillus ginseng microecological preparation (same as Example 5) was used as a feed additive to feed broiler chickens. The method was as follows: 120 broiler chickens with similar weights were selected, and the broiler chickens were numbered and divided into 4 groups, with 3 repetitions in each group. 10 per group. The 4 groups are:

Blank control group: fed basic diet;

Test group 1: fed basal diet and tetracycline;

Test group 2: fed basic diet and Bacillus ginseng bacteria agent (low dose: 500g, the number of viable bacteria in the bacterial powder is not less than 3×10 ⁸ CFU/g);

Test group 3: fed basic diet and Bacillus ginseng inoculant (high dose: 1500g, the number of viable bacteria in the bacterial powder is not less than 3×10 ⁸ CFU/g).

The experimental broilers were raised in cages, with free access to food and water, and a dedicated person was responsible for keeping records. The pre-feeding period is 7 days, and the official period is from 8 weeks to 12 weeks old, a total of 28 days. Feed intake, body weight, feed-to-meat ratio, and diarrhea were recorded every day during the official period. The results are shown in Table 3.

Table 3 Results of feeding broilers with Bacillus ginseng bacteria

It can be seen from Table 3 that Bacillus ginseng can improve feed utilization (the feed-to-meat ratio is reduced by 0.36), reduce the diarrhea rate of broilers by 5.76%, and can play a role in replacing antibiotics.

The above-described embodiments only describe preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. All deformations and improvements shall fall within the protection scope determined by the claims of the present invention.

sequence list

<110> Huazhong Agricultural University

<120> A strain of Bacillus ginseng with high production of organic acids and its application in microecological preparations

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1453

<212> DNA

<213> Artificial Sequence

<400> 1

tggcggcgtg ctataatgca agtcgagcga actgatgaag agcttgcttt tgatcagtta 60

gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactaggat aactccggga 120

aaccggggct aatactggat aacttttctc tccgcatgga gagagattga aagatggctt 180

cggctatcac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac 240

caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacattggga ctgagacacg 300

gcccaaactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360

gagcaacgcc gcgtgagtga agaaggtctt cggatcgtaa aactctgttg ttagggaaga 420

acaagtatcg ttcgaatagg gcggtacctt gacggtacct aaccagaaag ccacggctaa 480

ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttatggggcg 540

taaagcgcgc gcaggcggtc ttttaagtct gatgtgaaag cccacggctc aaccgtggag 600

ggtcattgga aactggaaga cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660

gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac 720

tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 780

cgtaaacgat gagtgctaag tgttagaggg tttccgccct ttagtgctgc agctaacgca 840

ttaagcactc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa ttgacggggg 900

cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960

cttgacatcc tctgacctcc ctagagatag ggccttcccc ttcgggggac agagtgacag 1020

gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080

gcaacccttg accttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac 1140

aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200

acgtgctaca atggatggta caaagggctg cgagaccgcg aggttaagcc aatcccataa 1260

aaccattctc agttcggatt gcaggctgca actcgcctgc atgaagctgg aatcgctagt 1320

aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380

caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac cttttggagc cagccgccga 1440

aggtgacaga gtg 1453

Claims (9)

1. Bacillus ginseng strainBacillus ginsengihumi) BG2204, which is characterized in that it has been preserved in the chinese collection of typical cultures, with the preservation number cctccc NO: m2022496.

2. A method for culturing the ginseng bacillus with high yield of lactic acid is characterized in that the ginseng bacillus is inoculated in a fermentation culture medium after being activated, and is cultured for 18 to 22 hours at a temperature of between 35 and 40 ℃, molasses, corn steep liquor and calcium carbonate are added for continuous culture, and the culture is performed for 20 to 24 hours at a temperature of between 40 and 50 ℃;

the components of the fermentation medium comprise 20g/L to 40g/L of sucrose, 5g/L to 10g/L of bean pulp, 5g/L to 10g/L of yeast extract powder, 0.5g/L to 1g/L of zinc sulfate and 0.5g/L to 1g/L of magnesium sulfate.

3. A high-density culture method of ginseng bacillus, characterized in that the ginseng bacillus described in claim 1 is subjected to activation culture, the obtained seed liquid is inoculated into a fermentation culture liquid for fermentation, and the fermentation culture liquid is prepared from the fermentation culture medium described in claim 2 and water according to 31.75g:1.5L, and adding supplementary carbon source and nitrogen source in batches by adopting a variable speed feeding method.

4. The high-density culture method according to claim 3, wherein the fermentation temperature is 35-40 ℃ and the fermentation period is 18-22 hours.

5. The high-density culture method according to claim 3, wherein the carbon source comprises sucrose; the nitrogen source comprises yeast extract powder and soybean meal in a mass ratio of 1:1.

6. A bacterial powder, which is obtained by centrifugally drying bacterial liquid obtained by fermenting the bacterial powder by the high-density culture method according to any one of claims 3 to 5.

7. A fermented feed, characterized in that the feed comprises the following raw materials in parts by weight: 60 parts of corn straw with 15% of water content, 1 part of 10 ten thousand U/g xylanase, 20 parts of fermented feed fermentation liquor and 50 parts of bran water;

the preparation method of the fermented feed fermentation liquid comprises the following steps: 120g of molasses, 30g of corn meal and 1L of bran water, boiling for 30min, then fixing the volume to 1L, sterilizing for 30min at 121 ℃, then inoculating 5% of seed liquid obtained by culturing the ginseng bacillus in claim 1 through strain activation liquid, and culturing for 22h at 37 ℃ at 180 rpm/min; the mass concentration of the bran water is 10%;

the fermented feed is prepared by uniformly mixing the raw materials, filling the mixture into a sealed bag, and carrying out anaerobic fermentation for 5 days at 37 ℃.

8. A microecological preparation containing the bacillus ginseng according to claim 1, which is characterized by comprising the following components in parts by weight: the fermented feed fermentation broth of claim 7, wherein the fermented feed comprises 2 parts of chitosan oligosaccharide, 1 part of trehalose and 3 parts of zeolite powder.

9. A Bacillus Ginseng strain according to claim 1Bacillus ginsengihumi) Use of BG2204 or the probiotic of claim 8 in aquaculture and feeding broiler chickens.