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

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

Technical Field

The application relates to the technical field of microecologics, in particular to a bacillus ginseng strain capable of producing organic acid at high yield and application of the bacillus ginseng strain in the microecologics.

Background

A microbial preparation (Probiotics, also called a viable bacterial preparation, microbial agent) is a viable microbial preparation made of normal microorganisms or substances that promote the growth of microorganisms. At present, the microecological preparation is widely applied to various fields of livestock breeding, agricultural planting, medical care, health food, environmental management and the like.

Bacillus and lactobacillus are the most commonly used microecological preparation strain types, and bacillus has good tolerance and can produce abundant enzymes; lactic acid bacteria metabolize to produce lactic acid, can inhibit the growth and reproduction of harmful bacteria, and has good microecological regulation effect. In the fields of livestock breeding, healthy foods and the like, the probiotics can be used for producing fermented foods, feeds and microecologics, can improve the feed utilization rate, increase the flavor of the foods, improve the intestinal microecological balance, enhance the immunity of hosts and improve the growth performance and the health level of the hosts. In the aspect of environmental management, bacillus and lactobacillus can also decompose organic matters, adjust water quality and microecological balance.

Grains, legumes, and oilseeds contain phytic acid, which can bind phosphorus, rendering 70% -80% of the phosphorus indigestible. This limits the use of these crops in feeds, especially in developing high-concentration diets for intensive livestock and poultry farming. Phytic acid is a special chemical derivative of hexainositol, whose six hydroxyl groups are bound to six phosphate residues. The phosphate residues are chemically active and 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, phytic acid, in addition to acting as a reservoir for phosphorus, incorporates a large proportion of trace elements, proteins, carbohydrates and amino acids, converting them into complex insoluble aggregates. Phytase (myo-inositol exakisporate phosporlase) is a special class of phosphomonoesterases which hydrolyses phytic acid to release phosphate. In the digestive tract of pigs, poultry and other monogastric animals, the yield of phytase is very limited. Most microbial phytases are intracellular enzymes, but fungi, enterobacteria and bacillus bacteria are capable of producing secreted phytases, which has driven the wide application of phytases in industry and agriculture.

AnnaA et al found a phytase encoding gene in Bacillus Ginseng M2.11 in 2015, revealing that the strain was able to secrete phytase. However, bacillus, especially ginseng bacillus, which can hydrolyze phytic acid and can produce organic acid at high yield is not reported.

Disclosure of Invention

The application aims to provide the ginseng bacillus with high yield of organic acid and the application thereof in the microecological preparation, and the ginseng bacillus is prepared into fermented feed and the microecological preparation through optimizing the fermentation culture and the acid production capacity thereof, and the effect of the ginseng bacillus in aquaculture and broiler chicken feeding is verified, so that a theoretical basis is provided for the development of the ginseng bacillus into the microecological preparation applied to the fields of livestock breeding, environmental management and the like.

In order to achieve the above object, the present application provides the following solutions:

the application provides a bacillus ginseng (Bacillus ginensis) BG2204 which is preserved in China Center for Type Culture Collection (CCTCC) NO: m2022496, the preservation address is Wuhan, university of Wuhan, and the preservation date is 2022, 04 and 26.

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

Further, 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.

The application also provides a high-density culture method of the ginseng bacillus, which comprises the steps of carrying out activation culture on the ginseng bacillus, inoculating the obtained seed liquid into a fermentation culture solution for fermentation, wherein the fermentation culture solution comprises 31.75g of fermentation culture medium and 31.75g of water: 1.5L, and adding supplementary carbon source and nitrogen source in batches 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, adding no carbon source, and keeping the rotating speed above the minimum rotating speed of 100 rpm/min; fermenting for 2-6 h, wherein the flow acceleration of the fed-batch carbon source is 3-6% of the total volume of fed-batch fermentation culture solution per hour, and the rotating speed is controlled to be 120-200 rpm/min; fermenting for 6-14 h, wherein the flow acceleration of the fed-batch carbon source is 5-10% of the total volume of fed-batch fermentation culture solution per hour, and the rotating speed is controlled to be 200-350 rpm/min; fermenting for 14-20 h, wherein the flow acceleration of the fed-batch carbon source is 5-10% of the total volume of fed-batch fermentation culture solution per hour, and the rotating speed is controlled to be 120-200 rpm/min; stopping feeding the carbon source in the final fermentation stage for 20-22 h;

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

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

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

The application also provides a fermented feed, which 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;

wherein, the fermented feed fermentation liquor is prepared by the following method: 120g of molasses, 30g of corn meal and 1L of bran water are mixed, boiled for 30min, fixed to 1L, sterilized for 30min at 121 ℃, then inoculated with 5% of ginseng bacillus seed liquid, and cultured 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 ℃.

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

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

The application discloses the following technical effects:

(1) The application separates and screens out a strain of ginseng bacillus with high yield of organic acid from distilled spirit yellow water, optimizes the culture medium and fermentation process, and the number of the optimized ginseng bacillus viable count reaches 3.5 multiplied by 10 ⁹ CFU/mL。

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

(3) The high-quality fermented feed containing the ginseng bacillus produced by the application contains organic acids such as lactic acid, acetic acid and the like. Through reasonable utilization of the industrial byproduct 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 application applies the bacillus of ginseng to aquaculture and feeding broiler chickens, obviously improves the absorption of crucian to feed and improves the immunity of the crucian; the diarrhea rate and the feed conversion ratio of the broiler chicken are obviously reduced, and the effect of replacing antibiotics is achieved. Provides a theoretical basis for the development of the microecological preparation applied to the fields of livestock breeding, environmental management and the like.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are 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 application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a colony morphology of Bacillus ginseng;

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

FIG. 3 is a gram of Bacillus Ginseng;

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

FIG. 5 is a diagram showing the optimal selection of the types and the addition amounts of carbon sources in the fermentation of the ginseng bacillus, wherein a is the growth condition of thalli under different carbon source types, b is the growth condition of thalli under different addition amounts by taking sucrose as a carbon source;

FIG. 6 is a diagram showing the optimized screening of the nitrogen source species in the fermentation of Bacillus ginseng;

FIG. 7 is a diagram showing the optimized selection of nitrogen source addition in the fermentation of Bacillus Ginseng, wherein a is the growth of thallus under different addition levels of magnesium sulfate, and b is the growth of thallus under different addition levels of zinc sulfate;

FIG. 8 is a diagram showing the optimal screening of inorganic salt types and addition amounts in fermentation of Bacillus ginseng;

FIG. 9 is a screen for optimizing fermentation conditions of Bacillus GinsengSelecting a graph, wherein a is different fermentation periods, and OD in bacterial liquid ₆₀₀ The measured value of (a) is the number of viable bacteria of the bacterial liquid under different initial fermentation pH values, c is the number of viable bacteria of the bacterial liquid under different fermentation temperatures, d is the number of viable bacteria of the bacterial liquid under different shaking bottle rotating speeds, e is the number of viable bacteria of the bacterial liquid under different inoculation amounts, and f is the number of viable bacteria of the bacterial liquid under different liquid loading amounts.

Detailed Description

Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.

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 application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 application 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 application. All documents mentioned in this specification are incorporated by reference 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 application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The reagents and materials used in the examples, unless otherwise specified, are commercially available from those skilled in the art.

Example 1 screening identification of Bacillus Ginseng

1. Screening of strains

Firstly, 0.5mL white spirit yellow water is taken, 10 is selected according to a dilution coating method ^-6 The dilution is coated on a flat plate, the coating amount is 100 mu L, the culture is carried out for 24 hours at 37 ℃, the strain with larger diameter which generates yellow transparent rings is selected for flat plate streaking and rescreening, and finally single colony is selected and inoculated on YPD liquid culture medium at 37 ℃ and 180rpm/min for enrichment culture.

2. Identification of species

2.1 morphological identification

The strain was streaked on YPD plates, and colonies of the strain were pale yellow in the middle, white in the edges, smooth in the surface, circular in shape, and about 2mm in colony diameter (FIG. 1).

Gram staining of the strain: the strain was observed to be gram positive by light microscopy oil microscopy and sporulated (FIG. 3).

2.2 identification of Phytase producing Capacity

The bacterial solutions were inoculated onto double-layer YPD plates to which 0.25g, 0.5g, 0.75g and 1.0g of calcium phytate were added, and the strains were observed to have the ability to hydrolyze calcium phytate, and the diameters of transparent circles were 23mm, 20mm, 19mm and 19mm, respectively (FIG. 2).

2.3 identification of organic acid production Capacity

The supernatant after centrifugation of the bacterial liquid was subjected to high performance liquid chromatography, the chromatographic column was Aminex HPX-87H (300 mm. Times.7.8 mm) for BIO-RAD, and part of the organic acids in the single sample could be separated at one time, and as can be seen from FIG. 4, lactic acid, acetic acid, propionic acid and other organic acids were produced in the bacterial liquid, wherein the lactic acid peak time was 13min, acetic acid peak time was 15min, propionic acid peak time was 18min, the peak of 13.406 in FIG. 4 was lactic acid, the peak of 15.701 was acetic acid, the peak of 18.524 was propionic acid, and the lactic acid content was calculated to be about 1g/L based on lactic acid standard curve y=253019x-5161 (where y is peak area, and x is lactic acid content).

2.4 molecular biological characterization

The bacterial strain is subjected to DNA extraction, amplified by a bacterial universal primer and then subjected to 16SrDNA sequencing analysis, so that the partial sequence of the 16srDNA gene is shown as SEQ ID NO. 1.

SEQ ID NO.1：

TGGCGGCGTGCTATAATGCAAGTCGAGCGAACTGATGAAGAGCTTGCTTTTGATCAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTAGGATAACTCCGGGAAACCGGGGCTAATACTGGATAACTTTTCTCTCCGCATGGAGAGAGATTGAAAGATGGCTTCGGCTATCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTATCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCTCCCTAGAGATAGGGCCTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAGACCGCGAGGTTAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGACAGAGTG。

The result shows that the similarity with the ginseng bacillus is highest and reaches 99.9% through NCBI sequence comparison, and the strain is determined to be the ginseng bacillus. The strain is sent to China center for type culture collection (China) for collection (China 4 months) for collection, and is classified and named as bacillus ginseng (Bacillus ginensis) BG2204, and the collection number is CCTCC NO: m2022496, the preservation address is Wuhan, university of Wuhan, china.

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

1. Strain

Bacillus Ginseng BG2204 isolated and identified in example 1.

2. Optimization of culture Medium and culture conditions

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

2.1 optimization of carbon Source species

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

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

2.2 optimization of Nitrogen Source species

Performing nitrogen source type optimization selection on the culture medium finally determined in the section 2.1, wherein the nitrogen source types comprise: tryptone, yeast extract powder, peptone, corn flour, ammonium sulfate, urea, ammonium chloride, sodium nitrate and soybean meal, wherein the initial addition amount is 10g/L; the number of viable bacteria after each nitrogen source addition culture was recorded. As is clear from FIG. 6, the number of viable bacteria after adding yeast extract powder, soybean meal and tryptone is the highest, so that the nitrogen source yeast extract powder, soybean meal and tryptone are selected for the subsequent composition and proportion optimization of the compound nitrogen source.

Optimizing the composition and proportion of the composite nitrogen source, wherein the selection proportion is respectively yeast extract powder: tryptone=1:1, 1:2, 2:1, soybean meal: yeast extract = 1:1, 1:2, 2:1, soybean meal: tryptone=1:1, 1:2, 2:1, and the number of viable bacteria after culturing under each addition condition was recorded, specifically as shown in table 1.

TABLE 1 optimization of Compound Nitrogen Source ratio

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

Optimizing the total addition amount of the optimal composite nitrogen source, and respectively selecting 5g/L, 10g/L, 15g/L, 20g/L, 25g/L and 30g/L for an addition amount optimization experiment; as is clear from FIG. 6, when the amount of the compound nitrogen source to be added is 20g/L, the number of viable bacteria is the highest, so that 20g/L is selected as the optimum amount of the compound nitrogen source to be added.

2.3 optimization of inorganic salt species

Performing optimal selection of inorganic salt types on the culture medium finally determined in the section 2.2, wherein the inorganic salt types comprise: potassium dihydrogen phosphate, sodium chloride, magnesium sulfate, calcium chloride or zinc sulfate, with initial addition amount of 1.0g/L; the number of viable bacteria after each inorganic salt addition culture was recorded. As can be seen from fig. 7, when the inorganic salts are magnesium sulfate and zinc sulfate, the number of viable bacteria is high, so that magnesium sulfate and zinc sulfate are selected as the optimal inorganic salts.

The optimal inorganic salt addition amounts were optimized, and the addition amount optimization experiments were performed by selecting 0.25g/L, 0.5g/L, 0.75g/L, 1.0g/L, and 1.5g/L, respectively, and as shown in FIG. 8, when the addition amounts of magnesium sulfate and zinc sulfate were 1.0g/L and 0.75g/L, the viable count was high, and therefore, 1.0g/L of magnesium sulfate and 0.75g/L of zinc sulfate were selected, respectively, as the optimal inorganic salt addition amounts.

2.4 optimization of fermentation conditions

The fermentation conditions (fermentation time, initial fermentation pH, fermentation temperature, rotation speed of a shaking table, inoculation amount and liquid loading amount) of the strain are optimized on the basis of the optimized carbon source, nitrogen source and inorganic salt optimized culture medium.

Preferred embodiments of the fermentation cycle are: the OD value is measured every 2h from 0 h; preferred embodiments of the initial fermentation pH are: 3.4, 5, 6, 7, 8, 9; the preferred embodiment of the temperature is: 25 ℃, 30 ℃,35 ℃,40 ℃, 45 ℃; the preferred embodiment of the rotation speed of the cradle is: 140rpm/min, 160rpm/min, 180rpm/min, 200rpm/min, 220rpm/min; preferred embodiments of the inoculum size are: 2%, 4%, 6%, 8%, 10%; the preferred embodiments of the liquid loading are: 25mL/250mL, 50mL/250mL, 75mL/250mL, 100mL/250mL, 125mL/250mL;

as can be seen from FIG. 9, the fermentation period is 18 h-22 h; the pH 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 3.5X10 ⁹ CFU/mL。

High-density fermentation of ginseng bacillus on 3.10L fermentation tank

The secondary seed medium composition was the same as the activation medium in example 2, and specifically: 20g/L glucose, 20g/L tryptone, 10g/L yeast extract; culturing at 37℃for 24h at 180 rpm/min.

The fermentation medium bottom water 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 and 1.0g of magnesium sulfate, and regulating the pH to 5.0-7.0.

Preparing a fed-batch carbon source: the volume of the fed-batch carbon source is 1.8L, the components are 400 g-500 g of sucrose, and the sterilization is carried out for 20min at 115 ℃.

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

3.1 fermentation culture

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

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

3.2 preparation of bacterial powder

Centrifuging the fermented bacterial liquid at 8000rpm/min for 15-20 min, collecting 140-180 g of bacterial cells, spray drying at 110-140 deg.c and 60-70 deg.c to obtain 14-18 g of bacterial powder.

3.3 results

The viable count of the finally obtained bacterial powder is 3 multiplied by 10 ¹⁰ CFU/g。

Comparative example 1

The method was similar to that of example 2, but the optimization procedure was performed in the absence of carbon source type and addition amount, and the viable count was measured after the fermentation was completed, as compared with example 2. The result is that: 1.3X10 ⁹ CFU/mL。

Comparative example 2

The method was similar to that of example 2, but in the optimization process, the nitrogen source type and the addition amount were optimized only in comparison with example 2, and the viable count was measured after the fermentation was completed. The result is that: 1.9X10 ⁹ CFU/mL。

Comparative example 3

The method of implementation is the same as that of example 2, but in the optimization process, compared with example 2,only the inorganic salt type and the addition amount are lack to optimize, and the viable count is measured after the fermentation is finished. The result is that: 1.1X10 times ⁹ CFU/mL。

Example 3 optimization of acid production Capacity of Bacillus Ginseng

1. Strains: same as in example 2

2. Culture medium:

(1) The strain activation liquid culture was performed under the optimized conditions of example 2, and after the activation culture, the lactic acid content in the bacterial liquid was measured.

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

The components of the fermentation medium are as follows: 20g/L to 40g/L of sucrose, 5g/L to 10g/L of soybean meal, 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. Culturing at 35-40 deg.c for 18-22 hr, adding 30-50 g/L molasses, corn pulp 5-10 g/L and calcium carbonate 5-10 g/L into the culture medium, culturing at 40-50 deg.c for 20-24 hr, and measuring lactic acid content.

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

Example 4 preparation of Bacillus Ginseng fermented feed

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

(2) Preparing a fermented feed fermentation liquid:

120g of molasses, 30g of corn meal, 1L of bran water (10% of bran) and water are added, the volume is fixed to 1L after the mixture is boiled for 30min, sterilization is carried out for 30min at 121 ℃, and then 5% of ginseng bacillus seed liquid (obtained after bacterial activation liquid culture) is inoculated for 22h at 37 ℃ and 180 rpm/min. Measuring the number of live bacteria 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:

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

After 5d, detecting each index in the fermented feed as follows: ginseng bacillus 5.4X10 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 mildew; and lactic acid can play a role in changing the environment in the intestinal tract and adjusting the balance of the intestinal tract flora in the intestinal tract of animals.

Example 5 application of Ginseng Bacillus bacteria agent in aquaculture

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

Fermenting the fermented feed broth (viable count 3×10) ⁸ CFU/mL), chitosan oligosaccharide, trehalose and lime powder are mixed, stirred uniformly and sprayed into a pond according to different addition amounts. The crucian cultured in the experiment was purchased from the university of agriculture market in chinese, martial arts.

Before the start of the experiment, fish were purchased and temporarily reared in the pond for 15 days as a pre-feeding period. 600 fish were then randomly harvested in the pond and divided into a control group, a low dose experimental group and a high dose experimental group (200 fish per group, average body weight: 0.52.+ -. 0.18 g, average body length: 3.44.+ -. 0.37 cm), each group being provided with three replicates. Culturing was performed in a pond.

The addition amount of the microecological preparation of the low-dose experimental group 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), control group was not added with microecologics. Daily weight gain, number of survivors were recorded in the formal period. The results are shown in Table 2.

Table 2 results of feeding crucian carp with a bacillus cereus inoculant

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

Example 6 application of Ginseng Bacillus inoculant in raising broiler chickens

The bacillus renieratus microecologics (same as in example 5) is used as a feed additive for feeding broiler chickens, and the method is as follows: 120 broilers with similar weights were selected, and the broilers were numbered and then divided into 4 groups of 3 replicates, 10. The 4 groups are respectively:

blank control group: feeding basic daily ration;

test 1 group: feeding basic ration and tetracycline;

test 2 group: feeding basic ration and Ginseng radix bacillus agent (low dosage: 500g, viable count of bacterial powder is not less than 3×10) ⁸ CFU/g)；

Test 3 group: feeding basic ration and Ginseng radix bacillus agent (high dosage: 1500g, viable count of bacterial powder is not less than 3×10) ⁸ CFU/g)。

The test broiler is kept in cages, free to eat and drink water, and special people are responsible for feeding records. The pre-feeding period is 7 days, and the formal period is 28 days from 8 weeks of age to 12 weeks of age of broiler chickens. The food intake, weight, feed conversion ratio, diarrhea were recorded daily during the normal period, and the results are shown in Table 3.

TABLE 3 results of broiler feeding of Ginseng Bacillus inoculant

As shown in Table 3, the Bacillus Ginseng has the effects of improving feed utilization rate (reducing feed conversion ratio by 0.36), reducing diarrhea rate of broiler chicken by 5.76%, and substituting antibiotics.

The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.

Sequence listing

<110> university of agriculture in China

<120> A strain of Bacillus Ginseng with high yield of organic acid and its application 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 (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.