CN113234631A

CN113234631A - Bacillus licheniformis and fermentation method and application thereof

Info

Publication number: CN113234631A
Application number: CN202110553317.8A
Authority: CN
Inventors: 代珍青; ***; 黎卓莹; 邓远钊
Original assignee: Guangdong Hinabiotech Co ltd
Current assignee: Guangdong Hinabiotech Co ltd
Priority date: 2021-05-20
Filing date: 2021-05-20
Publication date: 2021-08-10

Abstract

The invention provides a bacillus licheniformis, wherein the bacillus licheniformis is bacillus licheniformis B-1126, and the accession number is GDMCC NO: 61564, respectively; the invention screens and ferments the bacillus licheniformis B-1126 to obtain the fermentation liquor. The screened bacillus licheniformis is simple, has no toxic or side effect, has no residue, is green and safe, and the fermentation liquor obtained by fermenting the bacillus licheniformis contains higher secreted amylase, protease and cellulase, and can also secrete antibacterial substances, so that the bacillus licheniformis has obvious inhibiting effects on staphylococcus aureus and clostridium perfringens, and has no immune stress. In addition, the invention can solve the problems of low spore content and low spore yield in the bacillus licheniformis fermentation liquor, and has higher fermentation activity and application value.

Description

Bacillus licheniformis and fermentation method and application thereof

Technical Field

The invention relates to the technical field of microbial fermentation and microbial transformation, in particular to bacillus licheniformis and a fermentation method and application thereof.

Background

Antibiotic replacement is a hot problem in the current feed additive industry research. The probiotics without residue and pollution is used as one of the substitutes of antibiotics, and has great potential. The bacillus licheniformis is one of probiotics and has important effects in regulating intestinal microecological function, promoting beneficial flora proliferation, improving organism immunity, etc. The research shows that the bacillus licheniformis has strong inhibiting effect on various plant soil-borne pathogenic bacteria, such as phytophthora, tuberculosis, sclerotinia sclerotiorum, rice blast and the like, and the research also shows that the bacillus licheniformis is applied to the animal husbandry, because the fermentation culture components and the fermentation process of the bacillus licheniformis can influence the spore yield of the bacillus licheniformis fermentation liquor, the problems of low spore content and spore yield of the bacillus licheniformis fermentation liquor commonly exist in the prior art, so that the prepared bacillus licheniformis fermentation liquor has poor inhibiting effect on staphylococcus aureus and clostridium perfringens.

In summary, there still exist technical problems to be solved in the technical fields of microbial fermentation and microbial transformation.

Disclosure of Invention

Based on the above, in order to solve the problem of low spore content and spore yield in the bacillus licheniformis fermentation liquor in the prior art, the invention provides bacillus licheniformis, and the specific technical scheme is as follows:

a Bacillus licheniformis, wherein the Bacillus licheniformis is Bacillus licheniformis B-1126, and the deposit number thereof is GDMCC NO: 61564.

the invention also provides a fermentation method of the bacillus licheniformis, which comprises the following steps:

inoculating bacillus licheniformis on an LB culture medium for activation culture;

inoculating the activated bacillus licheniformis into a shake flask seed culture medium, and carrying out shake flask culture to obtain a first-stage seed solution;

coating the primary seed liquid on a screening plate for culturing for 24 hours, and then picking a single colony with a larger transparent ring on the screening plate;

inoculating the single colony in a secondary seed tank, and stirring and culturing to obtain a secondary seed solution;

inoculating the secondary seed liquid into a fermentation tank for fermentation culture to obtain fermentation liquid;

wherein the culture medium for fermentation culture is as follows: 25-35 g/L of corn starch, 8-12 g/L of molasses, 3-6 g/L of soybean meal, 3-6 g/L of peptone, 1-3 g/L of sodium chloride, 0.3-0.8 g/L of monopotassium phosphate, 1-3 g/L of dipotassium phosphate, 0.3-0.8 g/L of magnesium sulfate and 1-3 g/L of defoaming agent.

Further, the culture temperature of the screening plate was 36 ℃.

Further, the inoculation amount of the secondary seed liquid is 1% -5%.

Further, the culture temperature in the fermentation tank is 30-38 ℃; the culture time in the fermentation tank is 24-26 h.

Further, the pressure in the fermentation tank is 0.02-0.08 MPa, and the air flow introduced into the fermentation tank is 0.5-2.0 vvm.

Further, the activation culture is to streak and transfer the picked single colony into an LB culture medium, and culture for 10h-16h under the temperature condition of 30-36 ℃.

Further, the temperature in the secondary seed tank is 33-36 ℃, the stirring speed is 100-150 r/min, the ventilation volume is 0.3-0.6 vvm, the pressure is 0.03-0.05 Mpa, and the culture time is 24-26 h.

Furthermore, the total bacteria number of the fermentation liquor reaches more than 80 hundred million CFU/mL, the spore number reaches more than 76 hundred million CFU/mL, and the spore yield reaches more than 93 percent.

In addition, the invention also provides application of the fermentation liquor in inhibiting clostridium perfringens and staphylococcus aureus.

The screened bacillus licheniformis in the scheme is simple, has no toxic or side effect, no residue, is green and safe, and fermentation liquor obtained by fermenting the bacillus licheniformis contains higher secreted amylase, protease and cellulase, and can also secrete antibacterial substances, so that the bacillus licheniformis has obvious inhibiting effects on staphylococcus aureus and clostridium perfringens, and has no immune stress. In addition, the invention can solve the problems of low spore content and low spore yield in the bacillus licheniformis fermentation liquor, and has higher fermentation activity and application value.

Drawings

FIG. 1 is a schematic representation of the growth curve of Bacillus licheniformis B-1126 in example 1;

FIG. 2 is a schematic representation of the infection rate of Clostridium perfringens in the application test example;

FIG. 3 is a schematic illustration of the bacteriostatic action of Clostridium perfringens CVCC2027 by the fermentation broth prepared in example 8, nosiheptide, enrah, bacitracin zinc;

FIG. 4 is a schematic illustration of the bacteriostatic action of the fermentation broth, nosiheptide, enrah, bacitracin zinc prepared in example 8 on Clostridium perfringens CVCC 2038;

FIG. 5 is a schematic illustration of the bacteriostatic action of the fermentation broth, nosiheptide, enra, bacitracin zinc prepared in example 8 on clinical Clostridia of chicken origin;

FIG. 6 is a schematic illustration of the bacteriostasis of swine clinical Clostridium using the fermentation broth, nosiheptide, enrah, bacitracin zinc prepared in example 8.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to embodiments thereof. It should be understood that the detailed description and specific examples, while indicating the scope of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In one embodiment of the invention, the Bacillus licheniformis is Bacillus licheniformis B-1126(Bacillus licheniformis B-1126), and the deposit number thereof is GDMCC NO: 61564, respectively; deposited at the Guangdong province culture Collection in 2021, No. 3/17, on the following addresses: wulou of microbiological research institute experimental building of Guangdong province No. 100, Zhongluo, Guangzhou city; the classification is named as: bacillus licheniformis.

In one embodiment, a method for fermenting bacillus licheniformis is provided, which comprises the following steps:

In one embodiment, the screening plate is incubated at a temperature of 36 ℃.

In one embodiment, the secondary seed solution is inoculated in an amount of 1% to 5%.

In one embodiment, the culture temperature in the fermentation tank is 30-38 ℃; the culture time in the fermentation tank is 24-26 h.

In one embodiment, the pressure in the fermentation tank is 0.02MPa-0.08MPa, and the air flow introduced into the fermentation tank is 0.5vvm-2.0 vvm.

In one embodiment, the activated culture is to streak and transfer the picked single colonies into LB culture medium, and culture for 10h-16h under the temperature condition of 30-36 ℃.

In one embodiment, the temperature in the secondary seed tank is 33-36 ℃, the stirring speed is 100-150 r/min, the ventilation volume is 0.3-0.6 vvm, the pressure is 0.03-0.05 Mpa, and the culture time is 24-26 h.

In one embodiment, the total bacteria number of the fermentation liquid is more than 80 hundred million CFU/mL, the spore number is more than 76 hundred million CFU/mL, and the spore yield is more than 93%.

In one embodiment, the fermentation broth is used for inhibiting clostridium perfringens and staphylococcus aureus.

Embodiments of the present invention will be described in detail below with reference to specific examples.

Example 1:

bacillus licheniformis B-1126 is isolated from the intestine of sows. Specifically, the intestinal tract is taken out after the sow is dissected, the intestinal tract is extruded, the intestinal tract content is discharged, the intestinal tract is cut open, the inner wall of the intestinal tract is scraped by a sterile blade, and an intestinal tract inner wall sample is obtained. The intestinal lining samples were diluted with 10 times of phosphate buffer, 100 μ L of the dilution was pipetted onto BHI solid plates using a pipette gun, coated evenly with a coating rod, numbered, and repeated for 3 times. After being evenly coated, the mixture is placed in a super clean bench for 5-10min, so that the bacteria liquid on the surface of the culture medium is fully absorbed. The plate was placed upside down and cultured in a 30 ℃ incubator for 48 hours. Selecting bacterial colonies with different forms, inoculating the bacterial colonies on a common broth plate for separation and purification, and measuring the antiviral function of different bacteria to obtain a strain capable of resisting the virus of the freshwater cultured animals, which is named as B-116. Taking B-116 subjected to BHI solid culture medium purification culture, streaking and inoculating a single colony on a BUG identification plate, culturing for 16-24 h at 36 ℃, taking an inoculation liquid of a bacteria identification kit IF-A when the colony size is proper, wiping the outer wall of the tube, and putting the tube into a turbidity meter to adjust the reading to be 100% T; an appropriate amount of single colonies were dipped into the inoculum using a sterile cotton swab to allow the turbidimeter to read between 92% T and 98% T and the mixture was transferred to GEN III plates using an 8-well pipette in a volume of 100. mu.L per well. Loading the identification plate into a Biolog system for culture, automatically reading by the system, and finally outputting an identification result as the bacillus licheniformis to obtain the bacillus licheniformis B-116;

the bacillus licheniformis B-1126 is inoculated on an LB culture medium and cultured for 28h in a shaking flask at the temperature of 36 ℃, and the growth curve of the bacillus licheniformis is drawn, as shown in figure 1.

Example 2:

inoculating the bacillus licheniformis B-1126 on an LB culture medium, culturing for 16h at the temperature of 30 ℃, then collecting the activated bacillus licheniformis, washing the activated bacillus licheniformis with physiological saline, and then performing shake flask culture in a shake flask seed culture medium to obtain a first-stage seed solution; coating the primary seed solution on a screening plate, culturing for 24h at 36 ℃, and then picking a single colony with a large transparent ring on the screening plate; inoculating the single colony into a secondary seed tank with the temperature of 33 ℃, the stirring speed of 150r/min, the ventilation volume of 0.3vvm and the pressure of 0.03Mpa, and culturing for 22h under stirring to obtain a secondary seed solution.

Example 3:

inoculating the bacillus licheniformis B-1126 on an LB culture medium, culturing for 16h at the temperature of 36 ℃, then collecting the activated bacillus licheniformis, washing the activated bacillus licheniformis with physiological saline, and then performing shake flask culture in a shake flask seed culture medium to obtain a first-stage seed solution; coating the primary seed solution on a screening plate, culturing for 24h at 36 ℃, and then picking a single colony with a large transparent ring on the screening plate; inoculating the single colony into a secondary seed tank with the temperature of 36 ℃, the stirring speed of 150r/min, the ventilation capacity of 0.6vvm and the pressure of 0.05Mpa, and culturing for 24 hours under stirring to obtain a secondary seed solution.

Example 4:

inoculating B-1126 strains of Bacillus licheniformis to an LB culture medium, culturing for 16h at the temperature of 36 ℃, collecting activated Bacillus licheniformis, washing the activated Bacillus licheniformis with physiological saline, and performing shake flask culture in a shake flask seed culture medium to obtain a first-stage seed solution; coating the primary seed solution on a screening plate, culturing for 24h at 36 ℃, and then picking a single colony with a large transparent ring on the screening plate; inoculating the single colony into a secondary seed tank with the temperature of 36 ℃, the stirring speed of 150r/min, the ventilation capacity of 0.6vvm and the pressure of 0.05Mpa, and culturing for 26h under stirring to obtain a secondary seed solution.

Following the conditions of examples 2-4, 5 experiments were conducted in each example to obtain a summary of viable shake flask and spore count tests for B-1126, Bacillus licheniformis, and the results are shown in Table 1 below.

Table 1:

from the data analysis in table 1, it can be seen that: the bacillus licheniformis B-1126 has higher viable count and spore rate after being cultured for 24h-26h, wherein the highest viable count is more than 60 hundred million/mL when being cultured for 24h, the spore count is more than 59 percent, the spore rate is more than 95 percent, and the viable count and the spore rate begin to decline when being cultured for 26h, although the viable count and the spore rate are higher, therefore, the culturing time of the bacillus licheniformis B-1126 is preferably 24 h.

Example 5:

inoculating the second-stage seed liquid with the inoculation amount of 5% into a fermentation tank with the culture temperature of 36 ℃, the pressure of 0.08Mpa, the air flow of 2.0vvm and the volume of 50L for fermentation culture for 26h to obtain fermentation liquor, wherein the culture medium for the fermentation culture is as follows: 10g/L of corn starch, 2g/L of molasses, 1g/L of peptone, 0.1g/L of sodium chloride, 0.3g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.3g/L of magnesium sulfate and 1g/L of defoaming agent.

Example 6:

inoculating the second-stage seed liquid with the inoculation amount of 5% into a fermentation tank with the culture temperature of 36 ℃, the pressure of 0.08Mpa, the air flow of 2.0vvm and the volume of 50L for fermentation culture for 26h to obtain fermentation liquor, wherein the culture medium for the fermentation culture is as follows: 25g/L of corn starch, 8g/L of molasses, 3g/L of soybean meal, 3g/L of peptone, 1g/L of sodium chloride, 0.3g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.3g/L of magnesium sulfate and 1g/L of defoaming agent.

Example 7:

inoculating the second-stage seed liquid with the inoculation amount of 5% into a fermentation tank with the culture temperature of 36 ℃, the pressure of 0.08Mpa, the air flow of 2.0vvm and the volume of 50L for fermentation culture for 26h to obtain fermentation liquor, wherein the culture medium for the fermentation culture is as follows: 35g/L of corn starch, 12g/L of molasses, 6g/L of soybean meal, 6g/L of peptone, 3g/L of sodium chloride, 0.8g/L of dihydrogen phosphate, 3g/L of dipotassium hydrogen phosphate, 0.8g/L of magnesium sulfate and 3g/L of defoaming agent.

Example 8:

inoculating the second-stage seed liquid with the inoculation amount of 5% into a fermentation tank with the culture temperature of 36 ℃, the pressure of 0.08Mpa, the air flow of 2.0vvm and the volume of 50L for fermentation culture for 26h to obtain fermentation liquor, wherein the culture medium for the fermentation culture is as follows: 30g/L of corn starch, 10g/L of molasses, 5g/L of soybean meal, 5g/L of peptone, 2g/L of sodium chloride, 0.5g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate and 2g/L of defoaming agent.

The fermentation ability of B-1126 was judged by examining the total number of bacteria, the number of spores and the spore formation rate of the fermentation liquids obtained in examples 5 to 8, and the results are shown in Table 2 below.

Table 2:

as can be seen from the data analysis in Table 2, when the fermentation culture is carried out in the culture medium of the present application, a 50L fermentation tank can reach the total bacteria number of the fermentation broth of more than 80 hundred million CFU/mL, the spore number of more than 76 hundred million CFU/mL, and the spore yield of more than 94%, while the formula components in the culture medium are changed in example 5, so that the total bacteria number, the spore number and the spore yield are all lower than those in examples 6-8, therefore, the Bacillus licheniformis B-1126 of the present invention has excellent fermentation capacity under the optimized formula of the culture medium, and the obtained fermentation broth has significant total bacteria number, spore number and spore yield.

In vitro bacteriostasis test:

and (3) determination of a test solution: 1ml (100g/T) of the fermentation broth prepared in example 8 was taken.

The determination method comprises the following steps: antibacterial activity determination is performed by adopting a filter paper determination method, 0.5ml of bacterial suspension of clostridium perfringens CVCC2027 is inoculated on an NA plate by a coating method (the preparation method comprises the steps of boiling 1000ml of distilled water, adding 10g of glucose, 20g of agar, 3g of beef extract, 10g of peptone and pH 7), filter paper sheets of respectively soaked determination test solutions are taken by a pair of sterilization forceps and are horizontally placed in a culture dish containing test bacteria, each group is repeated for 3 times, and clear water is used as a blank control. The prepared dish was placed in a 30 ℃ incubator at a constant temperature, observed after 48 hours, and the diameter of the zone of inhibition was measured by the cross method and recorded as test 1. In the same manner, the fermentation liquid prepared in example 8 was tested for the inhibition of clostridium perfringens CVCC2038 (recorded as test 2), clostridium gallinarum clinical clostridium (recorded as test 3), clostridium suis clinical clostridium (recorded as test 4), staphylococcus aureus ATCC25923 (recorded as test 5), and staphylococcus aureus cmcc (b)26003 (recorded as test 6), and the results of 5 repeated inhibition zone diameter measurements are shown in table 3 below.

Table 3:

as can be seen from the analysis of in vitro bacteriostatic test data in Table 3, the fermentation liquid of the invention has significant inhibitory effect on Clostridium perfringens and excellent inhibitory effect on Staphylococcus aureus.

In addition, in the in vitro test, the related comparative tests were also performed, and the reagents of the test and the results of the test are shown in table 4 below and in fig. 3 to 6. Wherein the fermentation broth is the fermentation broth prepared in example 8, the nosiheptide, enra and bacitracin zinc are commercially available products, and specific components are not described in detail under the condition of consistent test conditions.

Table 4:

from the data in table 4 and the analysis in fig. 3-6, it can be seen that the fermentation broth of the present invention has significant inhibitory effect on clostridium.

Application test example:

adding the fermentation liquor obtained in the example 8 into basal feed of the sow to obtain mixed feed without antibiotic;

selecting 50 pregnant sows in a Guangxi pig farm, detecting the content of clostridium in excrement of the pregnant sows under the condition of not using the fermentation liquor, and feeding the test sows with the mixed feed containing the fermentation liquor after the detection is finished, wherein the addition amount is 200 g/t. And respectively collecting all test sows to respectively detect the content of clostridium perfringens after feeding mixed feed containing the fermentation liquor for 15 days.

The detection of the content of the clostridium perfringens is operated according to the instruction of a kit for rapidly detecting the clostridium perfringens on site, and the result is read once in 4 hours and the result is read once in 5 hours to be used for judging the infection condition of the clostridium perfringens of sows. If the content of clostridium perfringens in the excrement is higher, the clostridium perfringens bred in the same time in the culture process is more, the hydrogen sulfide produced by metabolism is more, the generated ferrous sulfide is correspondingly increased, and the time for producing the same amount of black precipitate bacteria is shorter. The content of clostridium perfringens in feces can be determined according to the time and amount of the black color generation starting during the culture process. The relationship between clostridium perfringens and color response is shown in table 5 below, the results of using the dressing containing the fermentation broth for the test sows are shown in table 6, and the clostridium perfringens infection rate of the test sows is shown in fig. 2.

Table 5:

table 6:

from the analysis in table 6, in 50 pregnant sows, 18 of clostridium perfringens are heavily infected, 24 of clostridium perfringens are lightly infected, and the number of clostridium perfringens in intestinal tract of organism is always 8, and the analysis in fig. 2 shows that: the infection rates are 36%, 48% and 16% respectively. All test pigs in the test are fed with the mixed feed containing 200g/t of the fermentation liquor for 15 days. Feces from test pigs were collected 15 days later to check for clostridium perfringens content. The result of secondary detection after 15 days of using the mixed material containing the fermentation liquid is that 3 clostridium perfringens are severely infected, 5 clostridium perfringens are lightly infected, the number of clostridium perfringens in the intestinal tract of the organism is normally 42, and the analysis of the graph in combination with figure 2 shows that the infection rates are respectively 6%, 10% and 84%, and specifically: in the test example, 50 pregnant sows are tested for the content of clostridium perfringens, the content of the clostridium perfringens in the severely infected pregnant sows is 36 percent and 48 percent respectively, and the content of the clostridium perfringens is only 16 percent normally. The sow with serious infection is detected again after the fermentation liquor is used for 15 days, so that the number of the sow is reduced from 18 to 3, the number of the sow is reduced by 15 in total, and the improvement rate reaches 83.33 percent; the number of slightly infected sows is reduced from 24 to 5, and the total number of slightly infected sows is reduced by 19, so that the improvement rate reaches 79.17 percent; the number of sows with normal clostridium perfringens in the organism is increased from 8 to 42, and 34 sows recover to be normal, and the treatment rate reaches 80.95 percent. The fermentation liquid can effectively reduce clostridium perfringens in pig intestinal tracts, improve the health state of animals, and obviously reduce the infection rate of clostridium perfringens, thereby having remarkable progress.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The bacillus licheniformis is bacillus licheniformis B-1126, and the bacillus licheniformis has a deposition number of GDMCC NO: 61564.

2. a fermentation method of Bacillus licheniformis, which is characterized by comprising the following steps:

3. The method for fermenting Bacillus licheniformis according to claim 2, characterized in that the cultivation temperature of the screening plate is 36 ℃.

4. The fermentation method of bacillus licheniformis according to claim 2, characterized in that the inoculation amount of the secondary seed liquid is 1-5%.

5. The method for fermenting Bacillus licheniformis according to claim 2, characterized in that, the culture temperature in the fermentation tank is 30-38 ℃; the culture time in the fermentation tank is 24-26 h.

6. The method for fermenting Bacillus licheniformis according to claim 5, wherein the pressure in the fermentation tank is 0.02MPa-0.08MPa, and the air flow rate in the fermentation tank is 0.5vvm-2.0 vvm.

7. The fermentation method of bacillus licheniformis according to claim 2, characterized in that the activation culture is streaked and transferred to LB culture medium for 10h-16h at 30-36 ℃.

8. The fermentation method of bacillus licheniformis according to claim 2, characterized in that the temperature in the secondary seed tank is 33-36 ℃, the stirring speed is 100-150 r/min, the ventilation volume is 0.3-0.6 vvm, the pressure is 0.03-0.05 Mpa, and the cultivation time is 24-26 h.

9. The method for fermenting Bacillus licheniformis according to claim 2, characterized in that the total number of bacteria in the fermentation liquid is more than 80 hundred million CFU/mL, the number of spores is more than 76 hundred million CFU/mL, and the sporulation rate is more than 93%.

10. Use of the fermentation broth of claim 2 for the inhibition of clostridium perfringens and staphylococcus aureus.