CN115074281A - Bacillus ginseng strain capable of highly producing organic acid and application thereof in microecological preparation - Google Patents

Abstract

The invention discloses a bacillus ginseng strain capable of highly producing organic acid and application thereof in a microecological preparation, and belongs to the technical field of microecological preparations. The bacillus ginseng (Bacillus ginsengihumi) BG2204 is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2022496, the preservation address is Wuhan, Wuhan university, and the preservation date is 26/04 in 2022. According to the invention, the bacillus ginseng is prepared into the fermented feed and the microecological preparation by fermentation culture and acid production capability optimization of the bacillus ginseng, the effect of the bacillus ginseng in aquaculture and broiler feeding is verified, and a theoretical basis is provided for the bacillus ginseng developed into the microecological preparation applied to the fields of livestock breeding, environmental management and the like.

Description

Bacillus ginseng strain capable of highly producing organic acid and application thereof in microecological preparation

Technical Field

The invention relates to the technical field of microecological preparations, in particular to a bacillus ginseng strain capable of highly producing organic acid and application thereof in microecological preparations.

Background

Microbial preparations (Probiotics, also known as viable bacteria preparations and microbial agents) are live microbial preparations prepared by using normal microorganisms or substances for promoting the growth of microorganisms. At present, the microecologics are widely applied to various fields such as livestock breeding, agricultural planting, medical care, health food, environmental management and the like.

Bacillus and lactic acid bacteria are the most common microbial preparation strain types, and the bacillus has good tolerance and can produce abundant enzymes; the metabolism of lactic acid bacteria produces lactic acid, which can inhibit the growth and reproduction of harmful bacteria and has good micro-ecological regulation effect. In the fields of livestock breeding, health food and the like, the probiotics can be used for producing fermented food, feed and microecological preparation, can improve the utilization rate of the feed, increase the flavor of the food, improve the microecological balance of intestinal tracts, enhance the immunity of hosts, and improve the growth performance and the health level of the hosts. In the aspect of environmental management, the bacillus and the lactic acid bacteria can also decompose organic matters, regulate water quality and balance microecology.

The grains, beans and oilseeds contain phytic acid which binds phosphorus, rendering 70-80% of the phosphorus indigestible. This limits the use of these crops in feed, particularly in developing high concentration daily rations for intensive livestock and poultry farming. Phytic acid is a specific chemical derivative of hexakis-inositol, whose six hydroxyl groups are bound to six phosphate residues. The phosphate residue is 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, besides acting as a phosphate reservoir, phytic acid also binds a significant portion of trace elements, proteins, carbohydrates and amino acids, converting them into a complex insoluble aggregate. Phytases (myo-inositol exakisporate phosphatases) are a special class of phosphomonoesterases that hydrolyze phytic acid to release phosphate. 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 enterobacter and bacillus genus produce secreted phytases, which promotes the widespread use of phytases in agriculture and industry.

AnnaA et al found the phytase encoding gene in B.ginseng M2.11 in 2015, revealing that this strain is capable of secreting phytase. However, the bacillus capable of hydrolyzing phytic acid and producing organic acid with high yield, especially the bacillus ginseng, is not reported.

Disclosure of Invention

The invention aims to provide a bacillus ginseng with high organic acid yield and application thereof in a microecological preparation, prepares the bacillus ginseng into a fermented feed and the microecological preparation by optimizing the fermentation culture and the acid production capacity of the bacillus ginseng, verifies the effect in aquaculture and broiler chicken feeding, and provides a theoretical basis for developing the bacillus ginseng into the microecological preparation to be applied to the fields of livestock breeding, environmental management and the like.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a bacillus ginseng (Bacillus ginsengihumi) BG2204 which is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2022496, the preservation address is Wuhan, Wuhan university, and the preservation date is 26/04 in 2022.

The invention also provides a culture method of the high-yield lactic acid ginseng bacillus, which comprises the steps of activating the ginseng bacillus, inoculating the activated ginseng bacillus into a fermentation culture medium, culturing for 18-22 h at 35-40 ℃, adding molasses, corn steep liquor and calcium carbonate for continuous culture, and culturing for 20-24 h at 40-50 ℃.

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

The invention also provides a high-density culture method of the ginseng bacillus, which comprises the steps of activating and culturing the ginseng bacillus, inoculating the obtained seed liquid into a fermentation culture solution for fermentation, wherein the fermentation culture solution is prepared by fermenting the seed liquid in a fermentation culture medium and water according to the weight ratio of 31.75 g: 1.5L of the mixture is obtained, and then a carbon source and a nitrogen source are supplemented in batch by adopting a variable-speed feeding method.

Further, when the volume of the fermentation culture solution is 1.5L, the specific feeding method comprises the following steps:

fermenting for 0-2 h, wherein no carbon source is fed, and the rotating speed is kept above the minimum rotating speed of 100 rpm/min; fermenting for 2-6 h, wherein the feeding speed of the feeding carbon source is 3-6% of the total volume of the feeding fermentation culture solution per hour, and the rotating speed is controlled to be 120-200 rpm/min; fermenting for 6-14 h, wherein the feeding speed of the feeding carbon source is 5-10% of the total volume of the feeding fermentation culture solution per hour, and the rotating speed is controlled at 200-350 rpm/min; fermenting for 14-20 h, wherein the feeding speed of the feeding carbon source is 5-10% of the total volume of the feeding fermentation culture solution per hour, and the rotating speed is controlled to be 120-200 rpm/min; stopping feeding the carbon source in the last fermentation stage for 20-22 h;

fermenting for 0-2 h without adding compound nitrogen source; fermenting for 2-6 h, wherein the feeding speed of the feeding composite nitrogen source is 1-3% of the total volume of the feeding fermentation culture solution per hour; fermenting for 6-14 h, wherein the feeding speed of the compound nitrogen source is 3-10% of the total volume of the fed-batch fermentation culture solution per hour; fermenting for 14-20 h, wherein the feeding speed of the compound nitrogen source is 1-3% of the total volume of the fed-batch fermentation culture solution per hour, and the dissolved oxygen content in the whole fermentation process is controlled to be more than 25-45%; and stopping feeding the compound nitrogen source in the final fermentation stage for 20-22 h.

Further, the fermentation temperature is 35-40 ℃, and the fermentation period is 18-22 h. .

Further, the fed-batch carbon source comprises 400 g-500 g of cane sugar, and the fed-batch volume is 1.8L; the fed-batch composite nitrogen source comprises 100-150 g of yeast extract powder and soybean meal in a mass ratio of 1:1, and the fed-batch volume is 3L.

The invention also provides bacterial powder obtained by the high-density culture method, wherein the bacterial powder is obtained by centrifugally drying fermented bacterial liquid.

The invention also provides a fermented feed, which comprises the following raw materials in parts by weight: 60 parts of corn straw with the water content of 15%, 1 part of xylanase with the water content of 10 ten thousand U/g, 20 parts of fermented feed fermentation liquor and 50 parts of bran water;

wherein the fermented feed fermentation broth is prepared by the following method: mixing molasses 120g, corn flour 30g and bran water 1L, boiling for 30min, diluting to 1L, sterilizing at 121 deg.C for 30min, inoculating 5% Bacillus Ginseng seed solution, and culturing at 37 deg.C and 180rpm/min for 22 h; the bran water has a mass concentration of 10%;

the fermented feed is prepared by uniformly mixing the raw materials, then putting the mixture into a sealed bag, and performing anaerobic fermentation for 5d at 37 ℃.

The invention also provides a microecological preparation containing the bacillus ginseng, which comprises the following components in parts by weight: 2 parts of fermented feed fermentation liquor, 2 parts of chitosan oligosaccharide, 1 part of trehalose and 3 parts of zeolite powder.

The invention also provides application of the bacillus ginseng or the microecological agent in aquaculture and broiler chicken feeding.

The invention discloses the following technical effects:

(1) the invention separates and screens the ginseng bacillus for high yield of organic acid from yellow water of white spirit, and the living bacteria number of the optimized ginseng bacillus reaches 3.5 multiplied by 10 through the optimization of culture medium and fermentation process ⁹ CFU/mL。

(2) The invention adopts a two-step fermentation method, and utilizes molasses, corn steep liquor, soybean meal and other raw materials to obviously improve the lactic acid yield of the bacillus ginseng, wherein the lactic acid yield reaches 2-4 g/L.

(3) The high-quality fermented feed containing the bacillus ginseng produced by the invention contains organic acids such as lactic acid, acetic acid and the like. By reasonably utilizing the industrial by-product molasses and the agricultural waste corn straw, the solid waste is optimally utilized, and the effects of energy conservation and emission reduction are achieved.

(4) The invention applies the ginseng bacillus to aquaculture and feeding of broiler chickens, obviously improves the absorption of crucian to feed, and improves the immunity of crucian; remarkably reduces the diarrhea rate and the feed conversion ratio of the broiler chicken, and achieves the effect of replacing antibiotics. Provides a theoretical basis for the application of the microecological preparation in the fields of livestock breeding, environmental management and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a morphological diagram of a colony of Bacillus ginseng;

FIG. 2 is a hydrolysis diagram of calcium phytate of Bacillus Ginseng;

FIG. 3 is a gram stain of Bacillus Ginseng;

FIG. 4 is a graph of lactic acid content of Bacillus ginseng;

FIG. 5 is a diagram of the optimization screening of the types and addition amounts of carbon sources in the fermentation of Bacillus Ginseng, wherein a is the growth of the cells under different carbon source types, and b is the growth of the cells under different addition amounts with sucrose as the carbon source;

FIG. 6 is a diagram of the type-optimized screening of nitrogen sources in Bacillus Ginseng fermentation;

FIG. 7 is a diagram of the nitrogen source addition amount optimization screening in the fermentation of Bacillus Ginseng, wherein a is the growth of bacteria under different addition amounts of magnesium sulfate, and b is the growth of bacteria under different addition amounts of zinc sulfate;

FIG. 8 is a diagram of the optimization screening of the types and addition amounts of inorganic salts in the fermentation of Bacillus Ginseng;

FIG. 9 is a diagram of the optimization and screening of the fermentation conditions of Bacillus Ginseng, wherein a is OD in the bacterial liquid in different fermentation periods ₆₀₀ B is the viable count of the bacteria liquid at different initial fermentation pH values, c is the viable count of the bacteria liquid at different fermentation temperatures, d is the viable count of the bacteria liquid at different shake flask rotation speeds, e is the viable count of the bacteria liquid at different inoculum sizes, and f is the viable count of the bacteria liquid at different liquid loading amounts.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in 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 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 present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Reagents and materials used in the examples were all those conventionally available to those skilled in the art without specific description.

Example 1 screening and identification of Bacillus Ginseng

1. Screening of strains

Firstly, 0.5mL of white spirit yellow water is taken, and 10 is selected according to a dilution coating method ^-6 And (3) coating a plate by dilution, culturing at 37 ℃ for 24h with the coating weight of 100 mu L, selecting a strain with a large diameter and a yellow transparent ring, carrying out plate streaking and rescreening, finally selecting a single colony, inoculating the single colony in a YPD liquid culture medium, and carrying out enrichment culture at 37 ℃ and 180 rpm/min.

2. Identification of strains

2.1 morphological identification

The strain was streaked on YPD plates, and the colony of the strain was pale yellow in color, white in edge, smooth and moist in surface, and circular, and had a diameter of about 2mm (FIG. 1).

Gram staining of the strain: the strain was observed to be gram-positive bacteria, distantly sporulating, by optical microscopy, oil microscopy (fig. 3).

2.2 identification of Phytase production ability

The bacterial suspension was inoculated on a double-layered YPD plate to which 0.25g, 0.5g, 0.75g, and 1.0g of calcium phytate were added, respectively, and the strain was observed to have the ability to hydrolyze calcium phytate, and the diameters of the transparent circles were 23mm, 20mm, 19mm, and 19mm, respectively (FIG. 2).

2.3 identification of the ability to produce organic acids

After the supernatant liquid after the bacterial liquid centrifugation is subjected to high performance liquid chromatography analysis, a chromatographic column is Aminex HPX-87H (300mm multiplied by 7.8mm) of BIO-RAD, the chromatographic column can separate part of organic acid in a single sample at one time, according to the graph of figure 4, organic acid such as lactic acid, acetic acid, propionic acid and the like is generated in the bacterial liquid, wherein the lactic acid peak-off time is 13min, the acetic acid peak-off time is 15min, the propionic acid peak-off time is 18min, the peak of 13.406 in the graph of figure 4 is lactic acid, the peak of 15.701 is acetic acid, the peak of 18.524 is propionic acid, and according to the lactic acid standard curve y, 253019x-5161 (wherein y is a peak area, and x is lactic acid content), the lactic acid content is calculated to be about 1 g/L.

2.4 molecular biological identification

The strain is subjected to DNA extraction, bacterial universal primer amplification and 16SrDNA sequencing analysis, and the partial sequence of the 16srDNA gene is shown as SEQ ID No. 1.

SEQ ID NO.1：

TGGCGGCGTGCTATAATGCAAGTCGAGCGAACTGATGAAGAGCTTGCTTTTGATCAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTAGGATAACTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGCATGGAGAGAGATTGAAAGATGGCTTCGGCTATCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCTCCCTAGAGATAGGGCCTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAGACCGCGAGGTTAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGACAGAGTG。

The result of NCBI sequence comparison shows that the similarity with the ginseng bacillus is the highest and reaches 99.9 percent, and the strain is determined to be the ginseng bacillus. The strain is delivered to China center for type culture collection for preservation at 26.4.2022, and is classified and named as Bacillus ginseng (Bacillus ginsengihumi) BG2204, and the preservation number is CCTCC NO: m2022496, the preservation address is Wuhan university, Wuhan, China.

Example 2 culture Medium, optimization of culture conditions and high-Density fermentation of Bacillus Ginseng

1. Bacterial strains

The bacillus ginseng BG2204 separated and identified in the example 1.

2. Optimization of culture media and culture conditions

Strain activation medium: 20g/L glucose, 20g/L tryptone and 10g/L yeast extract powder; culturing at 37 deg.C and 180rpm/min for 24 h.

2.1 optimization of the type of carbon Source

And (3) performing carbon source type optimization selection on the culture medium, wherein the carbon source types comprise: alpha-lactose, glucose, sucrose, soluble starch, D-xylose and beta-cyclodextrin, wherein the initial addition amount is 5-10 g/L; the number of viable bacteria after addition culture of each carbon source was recorded. As can be seen from FIG. 5, since the number of viable bacteria was the highest after sucrose was added, the optimum carbon source was sucrose.

Optimizing the addition of the optimal carbon source, and selecting 5g/L, 10g/L, 20g/L, 30g/L and 40g/L respectively to perform an addition optimization experiment; as is clear from FIG. 5, the optimum addition amount was 30g/L, since the number of viable cells was the highest when the addition amount was 30 g/L.

2.2 optimization of Nitrogen Source species

And (3) optimally selecting nitrogen source varieties on the finally determined culture medium in part 2.1, wherein the nitrogen source varieties comprise: tryptone, yeast extract powder, peptone, corn flour, ammonium sulfate, urea, ammonium chloride, sodium nitrate and soybean meal, wherein the initial addition amount is 10 g/L; the number of viable bacteria after the addition culture of each nitrogen source was recorded. As can be seen from FIG. 6, since the viable count was the highest after the addition of yeast extract powder, soybean meal and tryptone, the subsequent optimization of the composition and ratio of the composite nitrogen source was performed by selecting the nitrogen source yeast extract powder, soybean meal and tryptone.

Optimizing the composition and proportion of the composite nitrogen source, wherein the proportion is respectively that yeast extract powder: tryptone ═ 1:1, 1:2, 2:1, soybean meal: yeast extract powder 1:1, 1:2, 2:1, soybean meal: the number of viable cells cultured under each addition condition was recorded as tryptone ═ 1:1, 1:2, and 2:1, and the results are shown in table 1.

TABLE 1 Complex Nitrogen Source proportion optimization

As can be seen from table 1, when the soybean meal: when the yeast extract powder is 1:1, the viable count is highest, so the soybean meal: the yeast extract powder is 1:1 which is the composition and proportion of the compound optimal nitrogen source.

Optimizing the total addition of the optimal composite nitrogen source, and respectively selecting 5g/L, 10g/L, 15g/L, 20g/L, 25g/L and 30g/L to perform addition optimization experiments; as is clear from FIG. 6, when the amount of the complex nitrogen source added is 20g/L, the number of viable cells is the highest, and therefore 20g/L was selected as the optimum amount of the complex nitrogen source added.

2.3 optimization of the type of inorganic salt

The optimal selection of inorganic salt species was performed on 2.2 parts of the finalized medium, the inorganic salt species including: monopotassium phosphate, sodium chloride, magnesium sulfate, calcium chloride or zinc sulfate, wherein the initial addition amount is 1.0 g/L; the number of viable bacteria after incubation with each inorganic salt addition was recorded. As can be seen from fig. 7, when the inorganic salts were magnesium sulfate and zinc sulfate, the number of viable bacteria was high, and magnesium sulfate and zinc sulfate were selected as the optimum inorganic salts.

As can be seen from FIG. 8, when the amount of magnesium sulfate was 1.0g/L and the amount of zinc sulfate was 0.75g/L, the number of viable bacteria was high, and therefore, magnesium sulfate was 1.0g/L and zinc sulfate was 0.75g/L, respectively, which were the optimum amounts of inorganic salts.

2.4 optimization of fermentation conditions

And (3) optimizing the fermentation conditions (fermentation time, initial fermentation pH, fermentation temperature, shaking table rotating speed, inoculation amount and liquid loading amount) of the strains on the basis of the optimized culture medium of the carbon source, the nitrogen source and the inorganic salt.

The preferred embodiment of the fermentation cycle is: measuring the OD value every 2h from 0 h; preferred embodiments of the initial fermentation pH are: 3.4, 5, 6, 7, 8, 9; the preferred embodiments of the temperature are: 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃; the preferred embodiment of the rotational speed of the rocking platforms is: 140rpm/min, 160rpm/min, 180rpm/min, 200rpm/min, 220 rpm/min; the preferred embodiment of the inoculation amount is: 2%, 4%, 6%, 8%, 10%; the preferred embodiment of the liquid loading amount is as follows: 25mL/250mL, 50mL/250mL, 75mL/250mL, 100mL/250mL, 125mL/250 mL;

as can be seen from FIG. 9, when the fermentation period is 18h to 22 h; the pH value of the initial fermentation is 5-7; the fermentation temperature is 35-40 ℃; the rotating speed of the shaking table is 160 rpm/min-220 rpm/min; the inoculation amount is 2% -4%; when the liquid loading amount is 30mL/250 mL-100 mL/250mL, the number of viable bacteria is the highest and reaches 3.5 multiplied by 10 ⁹ CFU/mL。

High Density fermentation of Bacillus Ginseng in 3.10L fermenter

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

The bottom water in the horizontal fermentation of 10L fermentation tank contains: 1.5L of water, 20g of cane sugar, 5g of yeast extract, 5g of soybean meal, 0.75g of zinc sulfate and 1.0g of magnesium sulfate, and adjusting the pH value to 5.0-7.0.

Preparation of a fed-batch carbon source: the volume of the carbon source fed-batch is 1.8L, the component is 400 g-500 g of cane sugar, and the sterilization is carried out for 20min at 115 ℃.

Preparation of fed-batch nitrogen source: the volume of the nitrogen source fed-batch was 3L, and the nitrogen source contained 1.5L of yeast extract powder (100 g-150 g) and 1.5L of soybean meal (100 g-150 g) were sterilized at 115 ℃ for 20 min.

3.1 fermentation culture

Multi-stage fed-batch fermentation: inoculating the ginseng bacillus seed liquid prepared in the secondary seed culture medium into a 10L full-automatic fermentation tank filled with 1.5L of bottom water according to 3 percent (V/V) of the tank placing volume, wherein the fermentation temperature is 35-40 ℃, and the fermentation period is 18-22 h. Fermenting for 0-2 h, wherein no carbon source is fed, and the rotating speed is kept above the minimum rotating speed of 100 rpm/min; fermenting for 2-6 h, wherein the carbon source flow acceleration rate is 40-90 mL/h, and the rotating speed is controlled at 120-200 rpm/min; fermenting for 6-14 h, wherein the carbon source flow acceleration rate is 60-120 mL/h, and the rotating speed is controlled at 200-350 rpm/min; fermenting for 14-20 h, wherein the carbon source flow acceleration rate is 40-90 mL/h, and the rotating speed is controlled at 120-200 rpm/min; stopping feeding the carbon source for 20-22 h in the final fermentation stage

Fermenting for 0-2 h without adding compound nitrogen source; fermenting for 2-6 h, wherein the composite nitrogen source flow acceleration rate is as follows: 20mL/h to 50 mL/h; fermenting for 6-14 h, wherein the composite nitrogen source flow acceleration rate is as follows: 50mL/h-280 mL/h; fermenting for 14-20 h, wherein the composite nitrogen source flow acceleration rate is as follows: 40mL/h to 80mL/h, and the dissolved oxygen content in the whole fermentation process is controlled to be more than 25 to 45 percent; and stopping feeding the compound nitrogen source in the final fermentation stage for 20-22 h.

3.2 preparation of fungal powder

Centrifuging the fermented bacterial liquid at 8000rpm/min for 15-20 min, collecting 140-180 g of thalli, and performing spray drying at the air inlet temperature of 110-140 ℃ and the air outlet temperature of 60-70 ℃ to obtain 14-18 g of bacterial powder.

3.3 results

Finally, the product is processedThe obtained bacterial powder has viable count of 3 × 10 ¹⁰ CFU/g。

Comparative example 1

The implementation method is the same as that of example 2, but in the optimization process, compared with example 2, only the type and the addition amount of the carbon source are lacked, and the number of the live bacteria is determined after the fermentation is finished. The result is: 1.3X 10 ⁹ CFU/mL。

Comparative example 2

The method is the same as example 2, but the optimization process is only lack of nitrogen source species and addition amount optimization compared with example 2, and the number of viable bacteria is measured after fermentation is finished. The results were: 1.9X 10 ⁹ CFU/mL。

Comparative example 3

The implementation method is the same as that of the embodiment 2, but compared with the embodiment 2, the optimization process only lacks the inorganic salt species and the addition amount is optimized, and the number of the live bacteria is measured after the fermentation is finished. The result is: 1.1X 10 ⁹ CFU/mL。

Example 3 optimization of acid-producing ability of Bacillus Ginseng

1. The strain is as follows: same as example 2

2. Culture medium:

(1) the culture of the activated strain liquid was carried out under the conditions optimized in example 2, and the content of lactic acid in the strain liquid was measured after the activated culture.

(2) And transferring the activated bacterial liquid to a fermentation culture medium for continuous culture.

The fermentation medium comprises the following components: 20-40 g/L of cane sugar, 5-10 g/L of soybean meal, 5-10 g/L of yeast extract powder, 0.5-1 g/L of zinc sulfate and 0.5-1 g/L of magnesium sulfate. The culture temperature is 35 ℃ to 40 ℃, the culture is carried out for 18h to 22h, then 30g/L to 50g/L molasses, 5g/L to 10g/L corn steep liquor, 5g/L to 10g/L calcium carbonate and 40 ℃ to 50 ℃ are added into the culture medium, the culture is continued for 20h to 24h, and the content of the lactic acid is measured.

The lactic acid content determination in the above steps adopts high performance liquid chromatography. The measurement results show that: compared with the lactic acid content (1 g/L-2 g/L) in the activation medium, the content is improved by about two times, and reaches 2 g/L-4 g/L.

Example 4 preparation of fermented feed of Bacillus Ginseng

(1) The strain activation liquid medium was the same as that of example 2;

(2) preparing fermented feed fermentation liquor:

120g of molasses and 30g of corn flour, adding 1L of bran water (10% of bran) into the mixture, boiling the mixture for 30min, fixing the volume to 1L, sterilizing the mixture for 30min at 121 ℃, inoculating 5% of bacillus ginseng seed solution (obtained after culture of strain activation liquid), and culturing the mixture for 22h at 37 ℃ and 180 rpm/min. Measuring the viable count of the ginseng bacillus in the seed liquid to be 3 multiplied by 10 ⁸ CFU/mL；

(3) The preparation method of the fermented feed comprises the following steps:

according to parts by weight, 60 parts of corn straw (with the water content of 15%), 1 part of xylanase (with the water content of 10 ten thousand U/g), 20 parts of fermented feed fermentation liquor and 50 parts of bran water are mixed uniformly and then are filled into a sealed bag, and the mixture is subjected to anaerobic fermentation for 5 days at the temperature of 37 ℃ to obtain the bacillus ginseng fermented feed.

After 5d, detecting that each index in the fermented feed is respectively as follows: ginseng bacillus 5.4X 10 ⁹ CFU/g, lactic acid content 5.39%, acetic acid content 3.94%, acidity 9.76%. The increase of the acetic acid content in the fermented feed can effectively inhibit the growth of mould spores in the feed and effectively prevent the feed from mildewing; and the lactic acid can play a role in changing the environment in the intestinal tract and adjusting the balance of the gastrointestinal flora in the intestinal tract of animals.

Example 5 application of Bacillus Ginseng inoculum in aquaculture

Preparing a microecological preparation: the fermented feed prepared in the embodiment 4 comprises 2 parts of fermented feed broth, 2 parts of chitosan oligosaccharide, 1 part of trehalose and 3 parts of zeolite powder in parts by weight.

Fermenting the fermented feed fermentation liquid (viable count 3 × 10) ⁸ CFU/mL), mixing with chitosan oligosaccharide, trehalose and lime powder, stirring uniformly, and sprinkling into the pond according to different addition amounts. The crucian cultured in the experiment was purchased from the vegetable market of Huazhong agriculture university in Wuhan City.

Before the start of the experiment, fish were purchased and temporarily kept in the pond for 15 days as a pre-feeding period. Then, 600 fish were randomly collected in the pond and divided into a control group, a low dose test group and a high dose test group (200 fish per group, average body weight: 0.52 + -0.18 g, average body length: 3.44 + -0.37 cm), and each group was set to three replicates. The culture was carried out in a pond.

The addition amount of the low-dose experimental group microecological preparation is 250 g/mu (the viable count of the bacterial powder is not less than 3 multiplied by 10) ⁸ CFU/g), the addition amount of the high-dose experimental group microecological preparation is 500 g/mu (the viable count of the bacterial powder is not less than 3 multiplied by 10) ⁸ CFU/g), no probiotics were added to the control group. Weight gain and survival number were recorded daily during the official period. The results are shown in Table 2.

TABLE 2 result of feeding crucian carp with bacillus ginseng fungicide

As shown in Table 2, the Bacillus Ginseng preparation can increase the weight gain rate (by 336%) and the survival rate (by 7.14%). The microbial inoculum can improve the absorption of the crucian to the feed and can also improve the immunity of the crucian.

Example 6 application of Bacillus Ginseng inoculum in broiler feeding

The broiler chickens were fed with the bacillus ginseng microecological preparation (same as example 5) as a feed additive by the method: 120 broilers with similar body weights are selected, the broilers are numbered and divided into 4 groups, each group is repeated for 3, and each group is 10. The 4 groups were:

blank control group: feeding basic ration;

test 1 group: feeding basal diet and tetracycline;

test 2 groups: feeding basic ration and Bacillus Ginseng microbial inoculum (low dose: 500g, viable count of bacterial powder is not less than 3 × 10) ⁸ CFU/g)；

Run 3 groups: feeding basic ration and Bacillus Ginseng microbial inoculum (high dose: 1500g, viable count of bacterial powder is not less than 3 × 10) ⁸ CFU/g)。

The experimental broiler chickens are raised in cages and fed with water freely, and the special people are responsible for feeding records. The pre-feeding period is 7 days, and the formal period is from 8 weeks to 12 weeks of age of the broiler chicken for 28 days. The results of the daily recording of feed intake, body weight, feed conversion ratio and diarrhea during the official period are shown in Table 3.

TABLE 3 result of feeding Bacillus ginseng inoculant to broiler chicken

As can be seen from Table 3, the ginseng bacillus can improve the utilization rate of the feed (the feed-meat ratio is reduced by 0.36), reduce the diarrhea rate of the broiler chicken by 5.76% and replace antibiotics.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> university of agriculture in Huazhong

<120> bacillus ginseng strain capable of highly producing organic acid and application thereof in microecological preparation

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1453

<212> DNA

<213> Artificial Sequence (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 ttattgggcg 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 (10)

1. A strain of Bacillus ginseng (Bacillus ginsengihumi) BG2204 is characterized in that the Bacillus ginseng BG2204 has been preserved in China center for type culture collection with the preservation number of CCTCC NO: m2022496.

2. A method for culturing the ginseng bacillus with high yield of lactic acid is characterized in that the ginseng bacillus as claimed in claim 1 is activated and then inoculated in a fermentation culture medium, cultured for 18 h-22 h at 35-40 ℃, added with molasses, corn steep liquor and calcium carbonate for continuous culture, and cultured for 20 h-24 h at 40-50 ℃.

3. The culture method according to claim 2, wherein the fermentation medium comprises 20-40 g/L of sucrose, 5-10 g/L of soybean meal, 5-10 g/L of yeast extract powder, 0.5-1 g/L of zinc sulfate and 0.5-1 g/L of magnesium sulfate.

4. A method for culturing ginseng bacillus at high density, characterized in that the ginseng bacillus of claim 1 is activated and cultured, and the obtained seed liquid is inoculated into a fermentation culture solution for fermentation, wherein the fermentation culture solution is prepared by mixing the fermentation culture medium of claim 3 and water according to the weight ratio of 31.75 g: 1.5L of the mixture is obtained, and then a carbon source and a nitrogen source are supplemented in batch by adopting a variable-speed feeding method.

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

6. The high-density cultivation method according to claim 4, wherein the carbon source comprises sucrose; the compound nitrogen source comprises yeast extract powder and soybean meal in a mass ratio of 1: 1.

7. A fungal powder obtained by centrifugal drying of a bacterial liquid obtained by fermentation according to any one of claims 4 to 6 by the high-density culture method.

8. The fermented feed is characterized by comprising the following raw materials in parts by weight: 60 parts of corn straw with the water content of 15%, 1 part of xylanase with the water content of 10 ten thousand U/g, 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: mixing molasses 120g, corn flour 30g and bran water 1L, boiling for 30min, diluting to 1L, sterilizing at 121 deg.C for 30min, inoculating 5% Ginseng radix bacillus liquid, and culturing at 37 deg.C and 180rpm/min for 22 h; the bran water has a mass concentration of 10%;

the fermented feed is prepared by uniformly mixing the raw materials, then putting the mixture into a sealed bag, and performing anaerobic fermentation for 5d at 37 ℃.

9. A microecological preparation comprising the Bacillus ginseng of claim 1, which comprises the following components in parts by weight: the fermented feed of claim 8, which comprises 2 parts of fermented feed liquid, 2 parts of chitosan oligosaccharide, 1 part of trehalose and 3 parts of zeolite powder.

10. Use of the bacillus ginseng as claimed in claim 1 or the microecological agent as claimed in claim 9 for aquaculture and feeding broiler chickens.

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