CN114317347B - Bacillus coagulans and application thereof, composition and fermentation culture method of bacillus coagulans - Google Patents
Bacillus coagulans and application thereof, composition and fermentation culture method of bacillus coagulans Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application discloses bacillus coagulans and application, a composition and a fermentation culture method of bacillus coagulans, wherein the name of the bacillus coagulans is NJ, and the classification name is: bacillus coagulans, accession number is: CCTCC NO: M20211445, the preservation date is: 2021, 11 and 18 days, the preservation unit is: china center for type culture Collection; the active ingredients of the composition comprise bacillus coagulans NJ, and the composition is a feed additive, an animal feed, a food additive, a food or a probiotic; the fermentation process of the bacillus coagulans is to inoculate the bacillus coagulans into a novel fermentation culture medium for fermentation culture, and culture the bacillus coagulans under the conditions of 45-50 ℃ and pH of 5.5-6.0 and dissolved oxygen of not less than 20 percent, and has the advantages of high spore yield, high thallus concentration and low bacterial contamination rate.
Description
Technical Field
The application relates to the technical field of microbial fermentation, in particular to bacillus coagulans and application, a composition and a fermentation culture method of bacillus coagulans.
Background
Bacillus coagulans (Bacillus coagulans) belongs to the genus Bacillus, and is gram positive, sportive, and capable of forming endospores. Bacillus coagulans can grow in both aerobic and anaerobic conditions, but the amount of lactic acid produced by fermentation under anaerobic conditions is significantly higher than that produced by fermentation under aerobic conditions. The bacillus coagulans has lactic acid production characteristics of lactic acid bacteria and stress resistance characteristics of bacillus, so that the bacillus coagulans becomes unique spore probiotics and is widely applied to the fields of medicines, foods, livestock, aquaculture and the like.
The bacillus coagulans has a strong enzyme production system, for example, amylase, protease and other enzymes can be produced, after the bacillus coagulans is planted in intestinal tracts, various enzymes secreted by the bacillus coagulans in the growth and propagation process can promote organisms to absorb nutrient substances, so that the utilization rate of the nutrient substances is improved, but the performance difference of different strains of bacillus coagulans is large, for example, the enzyme production capacities of different strains of bacillus coagulans are different, and therefore, the bacillus coagulans with excellent comprehensive performance is obtained through screening. In addition, the existing fermentation production of bacillus coagulans has the defects of high bacterial contamination rate, low spore yield and low bacterial count, is not beneficial to industrial production and reduces the quality of products containing bacillus coagulans, so that research and development of a fermentation culture method suitable for bacillus coagulans is important for improving the fermentation level of bacillus coagulans, and particularly improving the spore conversion rate of bacillus coagulans.
Disclosure of Invention
Aiming at the defects of the prior art, the application provides bacillus coagulans, application thereof, a composition and a fermentation culture method of the bacillus coagulans.
The technical scheme of the application is as follows:
in a first aspect, the present application provides a bacillus coagulans, the bacillus coagulans being named NJ, under the classification name: bacillus coagulans, accession number is: CCTCC NO: M20211445, the preservation date is: 2021, 11 and 18 days, the preservation unit is: china center for type culture Collection.
In a second aspect, the present application provides the use of a Bacillus coagulans strain as described in the first aspect for the preparation of a feed additive, an animal feed, a food additive, a food or a probiotic.
In a third aspect, the present application provides a composition, the active ingredient of which comprises bacillus coagulans as described in the first aspect.
Further, the composition is a feed additive, an animal feed, a food additive, a food or a probiotic.
Further, the composition also comprises one or more auxiliary materials.
Further, the composition also includes one or more probiotic microorganisms.
In a fourth aspect, the application provides a bacillus coagulans fermentation medium, which comprises the following components in parts by mass: 20 to 30 parts of yeast extract, 20 to 30 parts of peptone, 2 to 8 parts of beef extract, 5 to 10 parts of fermented soybean meal and 4.0 to 12.0 parts of inorganic salt.
Further, the inorganic salt is selected from one or more of sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate and manganese sulfate.
Further, the bacillus coagulans fermentation medium comprises the following components in parts by weight: 20 to 30 parts of yeast extract, 20 to 30 parts of peptone, 2 to 8 parts of beef extract, 5 to 10 parts of fermented soybean meal, 3 to 6 parts of sodium acetate, 0.5 to 3 parts of dipotassium hydrogen phosphate, 0.2 to 1.0 parts of magnesium sulfate, 0.1 to 0.3 parts of manganese sulfate and 0.1 to 0.3 parts of ferric sulfate.
In a fifth aspect, the present application provides a fermentation culture method of bacillus coagulans, comprising the steps of: inoculating the seed liquid of Bacillus coagulans into the Bacillus coagulans fermentation medium according to any one of the fourth aspect, and culturing at 45℃to 50℃at pH 5.5 to 6.0 and dissolved oxygen of not less than 20%.
The application provides bacillus coagulans and application, a composition and a fermentation culture method of bacillus coagulans, which have the following technical effects:
compared with GDMCC1.421 bacillus coagulans, the NJ bacillus coagulans of the application has better probiotic properties, and is specifically expressed as follows: the NJ bacillus coagulans has ideal inhibition effects on clostridium perfringens CP-1, staphylococcus aureus S, salmonella pullorum SA083, streptococcus dysgalactiae KYB428, escherichia coli K88, staphylococcus epidermidis Sp, salmonella Sah, streptococcus suis SS-2 and streptococcus suis SS-9, while the GDMCC1.421 bacillus coagulans has no obvious inhibition effects on staphylococcus aureus S, staphylococcus epidermidis Sp, streptococcus suis SS-2, streptococcus suis SS-9, escherichia coli K88, salmonella Sah and streptococcus dysgalactiae KYB428, and the inhibition effects of GDMCC1.421 bacillus coagulans on clostridium perfringens CP-1 and salmonella SA083 are not as good as that of bacillus coagulans NJ, and moreover, the acid production capacity of the NJ bacillus coagulans is better than that of the GDMCC1.421 bacillus coagulans. Animal experiments show that compared with GDMCC1.421 bacillus coagulans, the NJ bacillus coagulans is more beneficial to improving various physiological indexes of the suckling piglets, such as weight, weight gain/feed intake and the like, and the diarrhea rate of the experimental piglets is remarkably reduced.
The fermentation culture method of bacillus coagulans in the application is to inoculate seed liquid of bacillus coagulans into a novel fermentation culture medium for fermentation culture, and combine specific fermentation process control, culture under the conditions of 45 ℃ to 50 ℃, pH of 5.5 to 6.0 and dissolved oxygen not lower than 20%, the fermentation transformation rate can reach more than 90%, when the fermentation scale is 2 tons of fermentation tanks, the concentration of thalli in fermentation liquor of the lower tank can reach 120 hundred million CFU/mL, the spore concentration can reach 80 hundred million CFU/mL, thereby effectively solving the problems of high bacterial contamination rate, low spore yield and low bacterial count existing in the traditional fermentation production of bacillus coagulans, and being beneficial to improving the quality of bacillus coagulans products.
Drawings
The technical solution and other advantageous effects of the present application will be made apparent by the following detailed description of the specific embodiments of the present application with reference to the accompanying drawings.
FIG. 1 is a graph showing the inhibitory effects of two strains of Bacillus coagulans, NJ and GDMCC1.421, on indicator strain CP-1 in the present application.
FIG. 2 is a graph showing the inhibitory effects of two strains of Bacillus coagulans, NJ and GDMCC1.421, on indicator bacteria S in the present application.
FIG. 3 is a graph showing the inhibitory effects of two strains of Bacillus coagulans NJ and GDMCC1.421 on indicator strain Sp in the present application.
FIG. 4 is a graph showing the inhibitory effects of two strains of Bacillus coagulans, NJ and GDMCC1.421, on indicator SS-2 in the present application.
FIG. 5 is a graph showing the inhibitory effects of two strains of Bacillus coagulans, NJ and GDMCC1.421, on indicator strain SS-9 in the present application.
FIG. 6 is a graph showing the inhibitory effects of two strains of Bacillus coagulans, NJ and GDMCC1.421, on indicator K88 in the present application.
FIG. 7 is a graph showing the inhibitory effects of two strains of Bacillus coagulans, NJ and GDMCC1.421, on indicator strain Sah according to the present application.
FIG. 8 is a graph showing the inhibitory effects of two strains of Bacillus coagulans, NJ and GDMCC1.421, on indicator SA083 in the present application.
FIG. 9 is a graph showing the inhibitory effect of two strains of Bacillus coagulans, NJ and GDMCC1.421, on indicator strain KYB428 in the present application.
FIG. 10 is a gram of the fermentation broth of example 1 of the present application.
FIG. 11 is a graph showing the change in the consumption of phosphoric acid during fermentation in example 2 of the present application.
FIG. 12 is a graph showing the change of dissolved oxygen during fermentation in example 2 of the present application.
FIG. 13 is a graph showing the pH change during fermentation in example 2 of the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application.
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. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the application.
The following description of the embodiments is not intended to limit the preferred embodiments. In addition, in the description of the present application, the term "comprising" means "including but not limited to". Various embodiments of the application may exist in a range of forms; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the application; it is therefore to be understood that the range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, a "20 to 30 parts yeast extract" should be considered to describe the specific disclosed subranges from 20 to 23 parts, from 23 to 25 parts, from 25 to 27 parts, from 27 to 30 parts, etc., as well as individual numbers within the stated ranges, e.g., 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts, and 30 parts, whichever is applicable. In addition, whenever a numerical range is referred to herein, it is meant to include any reference number (fractional or integer) within the indicated range.
Unless otherwise indicated, the starting materials and reagents used in the following examples are commercially available or may be prepared by methods known in the art.
1. The bacteriostasis experiment involved in the embodiment of the application is shown.
1.1 indicating bacteria involved in bacteriostasis experiments
Table 1 indicates the species, source, characteristics and culture conditions of the bacteria
Remarks: the media referred to in Table 1 were purchased from Qingdao sea Bo Biotechnology Co., ltd and used under the manufacturer's conditions.
1.2 antibacterial experiment equipment
Oxford cup: the inner diameter is 6.0 plus or minus 0.1mm, the outer diameter is 7.8 plus or minus 0.1mm, and the height is 10.0 plus or minus 0.1mm;
a biochemical incubator;
CR 22G refrigerated high speed centrifuge (Hitachi).
1.3 antibacterial experiment method
S a Preparing bacillus coagulans supernatant: inoculating Bacillus coagulans stored in glycerol pipe to MRS agar medium slant, culturing at 40deg.C for 24 hr, selecting a loop of colony growing on MRS medium slant, inoculating to MRS liquid medium, and culturing at 40deg.C for 200r/min until OD of the bacterial solution is reached 600 1.8, centrifuging the bacterial liquid obtained by culture at the rotating speed of 12000r/min for 5min, and collecting supernatant for later use;
S b indicator bacteria to be in logarithmic growth phase, OD thereof 600 The requirements of Table 2 are met, the culture medium is diluted to the use multiple and then coated on a corresponding culture medium plate, and three parallel samples are arranged for each indicator bacterium;
table 2 indicates the log phase OD of the bacteria 600 Using dilution factors
| Name of the name | Numbering device | Log phase OD 600 | Dilution factor | Taking amount |
| Clostridium perfringens | CP-1 | 0.86 | 10 -1 | 100μL |
| Staphylococcus aureus | S | 1.72 | 10 -3 | 100μL |
| Salmonella pullorum | SA083 | 1.19 | 10 -4 | 100μL |
| Streptococcus dysgalactiae | KYB428 | 1.13 | 10 -3 | 100μL |
| Coli bacterium | K88 | 1.15 | 10 -3 | 100μL |
| Staphylococcus epidermidis | Sp | 1.42 | 10 -4 | 100μL |
| Salmonella bacteria | Sah | 0.98 | 10 -4 | 150μL |
| Streptococcus suis (S.suis) | SS-2 | 0.39 | 10 -3 | 100μL |
| Streptococcus suis (S.suis) | SS-9 | 0.52 | 10 -3 | 100μL |
S c Placing a plurality of sterile oxford cups on a culture medium flat plate coated with indicator bacteria at equal intervals;
S d and respectively adding 0.15mL of bacillus coagulans supernatant into each oxford cup, then placing the bacillus coagulans supernatant at 4 ℃ for diffusion for 24 hours, placing the bacillus coagulans supernatant at 37 ℃ for culture for 24 hours, measuring the diameter of the inhibition zone (the diameter of the oxford cup is not deducted) by adopting a vernier caliper, calculating the average diameter value of all the inhibition zones on each flat plate, and photographing and recording.
2. Experimental description of acid generating Capacity involved in the examples of the present application
2.1 Medium involved in acid production Capacity experiments
Commercially available MRS broth medium and commercially available MRS agar medium.
2.2 Equipment involved in acid production Capacity experiments
A pH meter.
2.3 Experimental methods
Inoculating Bacillus coagulans stored in glycerol pipe to MRS agar medium slant, culturing at 40deg.C for 24 hr, selecting a loop of colony growing on MRS agar medium slant, inoculating to MRS liquid medium, and culturing at 40deg.C for 200r/min to obtain culture solution with thallus concentration of 1.56X10 8 CFU/mL, pH of the culture broth was measured with a pH meter.
3. Description of animal experiments in accordance with the examples of the application
3.1, laboratory animals
A plurality of 'Du Changda' suckling piglets with good health condition and close weight at 21 days are selected and randomly divided into four groups, each group is provided with two repeats, 15 piglets are repeated, the birth date difference between any two piglets is +/-1 day, and the weight difference between any two piglets is +/-0.05 kg. The immunization program is carried out by staff according to the routine, the feeding management mode and the environmental condition of each group are the same, and the experimental period is 21 days.
3.2 Experimental reagents
Physiological saline;
the mixed antibiotic solution consists of terramycin calcium, aureomycin, enramycin and deionized water, wherein the concentration of terramycin calcium is 100ppm, the concentration of aureomycin is 75ppm, and the concentration of enramycin is 10ppm;
a probiotic agent, which consists of bacillus coagulans to be tested and glucose, wherein the effective concentration of the bacillus coagulans to be tested is 1.0x10 9 CFU/g。
3.3 Experimental methods
During the experimental period, physiological saline is filled into each piglet in a control group, mixed antibiotic solution is filled into each piglet in a control group and probiotics are filled into each piglet in the experimental group, the dosage of the corresponding agent is 2mL for each piglet, the corresponding agent is filled into each piglet for 1 time per day, the time of each filling is kept consistent, the initial weight of each piglet is recorded, the weight and the health condition of each piglet are recorded every day, and 21-day average weight (Kg/head) of each piglet, the daily average weight gain (g/d) in the experimental period, the daily average feed intake (g/d), the body/feed intake (g/g) and the diarrhea rate (%) of each group are calculated. "average weight at 21 days" refers to the average weight at 21 days for all surviving piglets in each group, each group taking the average of two replicates, the average weight at 21 days for all surviving piglets in each replicate in each group being: overall weight/number of surviving piglets at 21 days of age; the term "daily average weight gain in the experimental period" means the average daily weight gain in the experimental period of all piglets which survived the experimental period in each group, and each group takes the average value of two replicates, and the calculation formula of the average daily weight gain in the experimental period of all piglets which survived the experimental period in each replicate in each group is as follows: total weight of all surviving piglets at 21-initial total weight of all surviving piglets)/(number of surviving piglets in experimental days); "daily average feed intake" refers to the average daily feed intake in the experimental period for all piglets in each group that survived the experimental period; "body weight gain/feed intake" refers to the ratio of daily average weight gain/daily average feed intake over the experimental period for all piglets in each group that survived the experimental period; "diarrhea rate" refers to the percentage of the number of diarrhea piglets in each group over the total number of piglets in each group during the experimental period (21 days).
The embodiment of the application provides bacillus coagulans, which is named as NJ and classified as: bacillus coagulans, accession number is: CCTCC NO: M20211445, the preservation date is: 2021, 11 and 18 days, the preservation unit is: china center for type culture collection, preservation address: university of martial arts in chinese.
The screening process of NJ is as follows:
s1, collecting Sichuan Dekang pig manure samples, mixing the pig manure samples with glass beads and sterile water to prepare sample suspensions, diluting and coating the sample suspensions on an MRS nutrient agar plate, respectively setting three parallel samples at each dilution of each sample suspension, and culturing the MRS nutrient agar plate at 40 ℃ for 48 hours after coating;
s2, picking white bacterial colonies with irregular edges on an MRS nutrient agar plate for separation and purification, and then adopting a 100-time oil mirror to carry out microscopic examination on the single bacterial colonies obtained after purification, and picking out bacterial strains which are pure bacteria and have spores and are rod-shaped in microscopic examination results, wherein the bacterial strains which accord with the description about morphological characteristics and physiological and biochemical indexes of bacillus coagulans in the eighth edition of the Berger' S bacteria identification manual and the common bacteria system identification manual are primarily identified as bacillus coagulans;
s3, carrying out 16S rDNA sequence identification on the strain preliminarily identified as bacillus coagulans in the step S2, and completing sequencing by Beijing qingke biotechnology limited company;
s4, performing bacteriostasis experiments, acid production capability experiments and animal experiments on 6 strains of bacillus coagulans with 16S rDNA sequence identification results, and screening to obtain a strain of bacillus coagulans with excellent comprehensive performance, wherein the strain is named NJ;
bacillus coagulans with the number of GDMCC1.421 of China general microbiological culture collection center is selected as an experimental strain, and NJ and GDMCC1.421 are respectively subjected to a bacteriostasis experiment, an acidogenesis capability experiment and an animal experiment to compare the bacteriostasis capability, the acidogenesis capability and the probiotics capability to animals of the NJ and the GDMCC1.421, and the results are shown in the following tables 3 to 8.
For bacteriostasis experiments, fig. 1 to 9 show bacteriostasis effect graphs of two strains of bacillus coagulans of NJ and GDMCC1.421, and the bacteriostasis experiment results are shown in table 3 below:
table 3 bacteriostasis test results of two Bacillus coagulans strains NJ and GDMCC1.421
As can be seen from FIGS. 1 to 9 and Table 3, bacillus coagulans NJ has ideal inhibitory effects on clostridium perfringens CP-1, staphylococcus aureus S, staphylococcus epidermidis Sp, streptococcus suis SS-2, streptococcus suis SS-9, escherichia coli K88, salmonella Sah, salmonella pullorum SA083 and Streptococcus dysgalactiae KYB428, whereas Bacillus coagulans GDMCC1.421 has no obvious inhibitory effects on Clostridium perfringens S, staphylococcus epidermidis Sp, streptococcus suis SS-2, streptococcus suis SS-9, escherichia coli K88, salmonella Sah and Streptococcus dysgalactiae KYB428, and Bacillus coagulans GDMCC1.421 has inhibitory effects on Clostridium perfringens CP-1 and Salmonella SA083, but GDMCC 1.Bacillus coagulans has no inhibitory effects on Clostridium perfringens CP-1 and Salmonella SA083 as much as Bacillus coagulans NJ.
The results of the acid-producing ability experiments of two strains of Bacillus coagulans, NJ and GDMCC1.421, are detailed in Table 4 below:
TABLE 4 acid production ability test results of two Bacillus coagulans strains NJ and GDMCC1.421
| Bacillus coagulans name | pH value of |
| NJ | 4.61 |
| GDMCC1.421 | 5.25 |
As can be seen from Table 4, the acid generating capacity of NJ is significantly better than GDMCC1.421.
Animal experiment results of two strains of Bacillus coagulans, NJ and GDMCC1.421 are shown in Table 5 below:
table 5 Table of results of animal experiments with two Bacillus coagulans strains NJ and GDMCC1.421
As can be seen from table 5, the average weight of 21 days old, the average daily weight gain during the experimental period, the average daily feed intake during the experimental period, and the average body weight gain/feed intake of the suckling piglets in the experimental group and the experimental group were higher than those in the control group and the control group, and the diarrhea rate of the suckling piglets in the experimental group was lower than those in the control group, the control group and the experimental group, and the diarrhea rate of the suckling piglets in the experimental group was higher than that in the control group. Each index of the suckling piglet in the experiment group I is superior to that of the suckling piglet in the experiment group II, and the beneficial effect of NJ is fully demonstrated to be superior to GDMCC1.421.
The embodiment of the application also provides application of the bacillus coagulans NJ in preparing feed additives, animal feeds, food additives, foods or probiotics.
The embodiment of the application also provides a composition, and the active ingredients of the composition comprise bacillus coagulans NJ. It will be appreciated that Bacillus coagulans NJ may be present in the composition in the form of: pure bacillus coagulans NJ, bacillus coagulans NJ fermentation culture centrifugation obtained culture solution or dried product thereof, bacillus coagulans NJ fermentation culture centrifugation obtained thallus or dried product thereof. The composition may be a liquid or a solid, for example, the composition is in the form of a tablet, granule, powder, spray, suspension, emulsion, capsule or paste.
In some embodiments of the application, the composition is a feed additive, an animal feed, a food additive, a food or a probiotic. When the composition is a feed additive or an animal feed, the composition is fed to animals including but not limited to livestock such as pigs, chickens, cattle and sheep, has a probiotic function similar to that of an antibiotic feed, has no side effect of the antibiotic feed, can regulate microecological balance in the intestinal tract of the animal to improve the immune function of the organism, and has the effects of providing nutritional factors, promoting the digestion and absorption of nutritional substances, preventing or assisting in treating digestive system diseases (such as diarrhea) and promoting the gastrointestinal development of young animals, thereby being beneficial to the growth and development of the animals. When the composition is a food additive or food, it is mainly suitable for human beings and has a probiotic effect.
In some embodiments of the application, the composition further comprises one or more excipients. The auxiliary materials are additional components used in the production of the composition and the formulation of the prescription, and have important functions of shaping, protecting the active components, improving stability, solubilization, dissolution assistance, slow release and the like, so that the composition achieves a certain shelf life and bioavailability, thereby improving the safety and effectiveness of the composition, and the auxiliary materials comprise, but are not limited to, excipients, diluents, fillers, solvents, supporting agents, premixing agents, disintegrating agents, surfactants, adsorption carriers and the like. When the composition is used in the preparation of a feed, the adjuvant may be any conventional adjuvant in the field of animal feed science, the choice of adjuvant will depend on the mode of use of the composition. When the composition is applied to the preparation of a food, the adjunct may be a food science acceptable adjunct, such as a flavoring agent, a sweetener, etc.
In some embodiments of the application, the composition further comprises one or more probiotic microorganisms, for example: bifidobacteria, lactobacilli, and the like.
The embodiment of the application also provides a bacillus coagulans fermentation medium, which comprises the following components in parts by mass: 20 to 30 parts of yeast extract, 20 to 30 parts of peptone, 2 to 8 parts of beef extract, 5 to 10 parts of fermented soybean meal and 4.0 to 12.0 parts of inorganic salt.
In the bacillus coagulans fermentation medium, no sugar carbon source is required to be added, and the sugar carbon source comprises, but is not limited to, glucose, sucrose, molasses, starch, corn flour, sweet potato powder and the like.
In some embodiments of the application, the inorganic salt is selected from one or more of sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, and manganese sulfate.
In some embodiments of the application, the bacillus coagulans fermentation medium comprises, in parts by weight: 20. yeast extract 20-30 parts, peptone 20-30 parts, beef extract 2-8 parts, fermented soybean meal 5-10 parts, sodium acetate 3-6 parts, dipotassium hydrogen phosphate 0.5-3 parts, magnesium sulfate 0.2-1.0 parts, manganese sulfate 0.1-0.3 parts and ferric sulfate 0.1-0.3 parts.
The embodiment of the application also provides a fermentation culture method of bacillus coagulans, which comprises the following steps: inoculating seed liquid of bacillus coagulans into the bacillus coagulans fermentation medium according to any one of the embodiments of the application, and culturing under the conditions of 45-50 ℃ and pH of 5.5-6.0 and dissolved oxygen not lower than 20%.
The fermentation culture method of bacillus coagulans in the embodiment of the application is applicable to a shake flask fermentation stage, a small test stage, a pilot test stage, a large tank trial production stage and an industrial production stage, and fermentation scales include, but are not limited to, a 30L fermentation tank, a 100L fermentation tank, a 500L fermentation tank, a 2 ton fermentation tank, a 10 ton fermentation tank and the like, and a person skilled in the art can prepare seed liquid step by step according to the fermentation scales by relying on own mastered professional knowledge.
The following examples are given to illustrate the fermentation culture method and technical effects of Bacillus coagulans of the present application in detail by way of specific examples and comparative examples, and are not intended to limit the present application in any way.
Example 1
The present example provides a fermentation culture method of Bacillus coagulans NJ, wherein the fermentation scale is a 2 ton fermenter.
1. Preparation of seed liquid
(1) Seed activation
Taking the bacillus coagulans NJ stored in the glycerol pipe at the temperature of minus 80 ℃, sucking 1.0mL of bacterial liquid after the bacillus coagulans NJ glycerol pipe is completely melted, inoculating the bacillus coagulans NJ into 50mL of MRS liquid culture medium, placing the bacillus coagulans in a 45 ℃ and 250r/min condition, shake-flask culturing for 15h, then picking out bacterial liquid, streaking the bacterial liquid on a MRS culture medium flat plate, and placing the bacterial liquid in 45 ℃ for anaerobic culturing for 24h to obtain single bacterial colony of the bacillus coagulans NJ.
(2) Preparation of first seed liquid
The inoculating loop takes a loop of bacillus coagulans NJ single colony growing on an MRS culture medium flat plate, disperses in 2mL of physiological saline, then inoculates in 50mL of MRS liquid culture medium, and places the culture medium in a shake flask at 45 ℃ and 250r/min for 12h for standby.
(3) Preparing secondary seed liquid
Transferring the first-stage seed liquid into MRS liquid culture medium according to the inoculation amount of 2% by volume, and shaking and culturing at 45deg.C under 250r/min for 12 hr.
(4) Preparation of seed tank culture solution
A 100L fermentation tank is selected as a seed tank, a seed culture medium is added according to 60 percent of the volume of the tank, a polyether defoamer of 2 per mill (volume ratio, V/V) is added, the pH is regulated to 6.3 by sodium hydroxide, then sterilization is carried out for 20min at 121 ℃, cold water is introduced into a jacket after sterilization is finished so as to rapidly cool down, and sterile air is introduced into the tank for pressure maintenance of 0.05Mpa. Wherein, the seed culture medium consists of 20g/L yeast extract powder, 20g/L peptone, 5g/L beef extract, 5.0g/L sodium acetate, 2.0g/L dipotassium hydrogen phosphate, 1.0g/L magnesium sulfate, 0.5g/L calcium carbonate and 0.2g/L manganese sulfate according to the parts by weight, and the pH value of the seed culture medium is regulated to 6.3.
When the pot temperature of the seed pot is cooled to 47 ℃, inoculating a secondary seed solution according to the inoculation amount with the volume ratio of 1%, wherein the initial aeration ratio is 0.5vvm, maintaining the pot pressure at 0.05Mpa, controlling the temperature at 47 ℃, starting fermentation at the lowest stirring rotation speed, maintaining dissolved oxygen at not lower than 20% in the whole fermentation process, culturing until the transformation rate reaches more than 80%, and then carrying out water bath for 15min at 85 ℃ to obtain the seed pot culture solution.
2.2 ton fermentation tank fermentation production
(1) Fermentation medium
The fermentation medium consists of the following components:
20g/L yeast extract, 20g/L peptone, 2g/L beef extract, 5g/L fermented soybean meal, 3g/L sodium acetate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.5g/L calcium chloride, 0.1g/L manganese sulfate and 0.1g/L ferric sulfate.
Sodium hydroxide is used to adjust the pH of the fermentation medium to 6.3.
(2) 2 ton fermentation tank actual elimination
Adding a fermentation culture medium according to 60% of the volume of the tank, adding a polyether defoamer in an amount of 2 per mill (volume ratio, V/V), adjusting the pH to 6.3 by sodium hydroxide, introducing cold water into the jacket after sterilization is finished so as to rapidly cool down, and introducing sterile air into the tank for maintaining the pressure to 0.05Mpa.
(3) Fermentation control
And (3) when the pot temperature is cooled to 48 ℃, inoculating the seed pot culture solution into the pot according to the inoculation amount of 5% by volume, wherein the initial aeration ratio is 0.5vvm, maintaining the pot pressure at 0.05Mpa, controlling the temperature at 47 ℃, starting fermentation at the lowest stirring rotation speed, feeding 50% phosphoric acid to control the fermentation pH to be 5.9, maintaining the dissolved oxygen to be not lower than 20% in the whole fermentation process, and culturing until the transformation rate reaches more than 80% (shown in figure 10).
In this example, the fermentation broth from the lower tank was diluted and spread on MRS medium plates, three replicates were set for each dilution, and after 24 hours of stationary culture at 40℃the fermentation broth from the lower tank was counted, and the concentration of the bacterial cells in the fermentation broth from the lower tank was found to be 60 hundred million CFU/mL and the concentration of the spores was found to be 40 hundred million CFU/mL.
Example 2
The present example provides a fermentation culture method of Bacillus coagulans NJ, wherein the fermentation scale is a 30L fermenter. The specific operation process is as follows: the primary seed liquid and the secondary seed liquid are prepared and obtained according to the method in the example 1, the secondary seed liquid is inoculated into a 30L fermentation tank (the liquid loading amount is 16L, the fermentation medium is the same as the example 1, the actual elimination method of the fermentation tank is carried out according to the example 1), the initial aeration ratio is 0.5vvm, the tank pressure is maintained at 0.05Mpa, the temperature is controlled at 47 ℃, the fermentation is started at the lowest stirring rotation speed, 50% phosphoric acid is fed to control the fermentation pH to be 5.9 (the consumption of phosphoric acid in the fermentation process is shown in figure 11), the dissolved oxygen is maintained to be not lower than 20% in the whole fermentation process, the dissolved oxygen and the pH are detected at regular time, the fermentation can be carried out until the transformation rate reaches more than 80%, the dissolved oxygen change condition in the whole fermentation process is shown in figure 12, and the pH change condition is shown in figure 13.
In this example, the fermentation broth from the lower tank was diluted and spread on MRS medium plates, three replicates were set for each dilution, and after 24 hours of stationary culture at 40℃the fermentation broth from the lower tank was counted, and the concentration of the bacterial cells in the fermentation broth from the lower tank was found to be 80 hundred million CFU/mL, and the concentration of the spores was found to be 60 hundred million CFU/mL.
Example 3
The present example provides a fermentation culture method of Bacillus coagulans NJ, wherein the fermentation scale is a 2 ton fermenter. Compared with the fermentation process of example 1, the fermentation process of this example is different in that: the fermentation media are not identical.
The fermentation medium of this example is: 25g/L yeast extract, 25g/L peptone, 5g/L beef extract, 8g/L fermented soybean meal, 5g/L sodium acetate, 2g/L dipotassium hydrogen phosphate, 0.5g/L magnesium sulfate, 0.5g/L calcium chloride, 0.2g/L manganese sulfate and 0.2g/L ferric sulfate.
In this example, the fermentation broth from the lower tank was diluted and spread on MRS medium plates, three replicates were set for each dilution, and after 24 hours of stationary culture at 40℃the fermentation broth from the lower tank was counted, and the concentration of the bacterial cells in the fermentation broth from the lower tank was found to be 80 hundred million CFU/mL, and the concentration of the spores was found to be 60 hundred million CFU/mL.
Example 4
The present example provides a fermentation culture method of Bacillus coagulans NJ, wherein the fermentation scale is a 2 ton fermenter. Compared with the fermentation process of example 1, the fermentation process of this example is different in that: the fermentation media are not identical.
The fermentation medium of this example is: 30g/L yeast extract, 30g/L peptone, 8g/L beef extract, 10g/L fermented soybean meal, 6g/L sodium acetate, 3g/L dipotassium hydrogen phosphate, 1g/L magnesium sulfate, 0.5g/L calcium chloride, 0.3g/L manganese sulfate and 0.3g/L ferric sulfate.
In this example, the fermentation broth from the lower tank was diluted and spread on MRS medium plates, three replicates were set for each dilution, and after 24 hours of stationary culture at 40℃the fermentation broth from the lower tank was counted, and the concentration of the bacterial cells in the fermentation broth from the lower tank was 120 hundred million CFU/mL and the concentration of the spores was 80 hundred million CFU/mL.
Comparative example 1
The present comparative example provides a fermentation process of bacillus coagulans, wherein the fermentation scale is a 2 ton fermenter. The fermentation process of this comparative example is different from the fermentation process of example 1 in that: the fermentation media are not identical.
The fermentation medium of this comparative example was: 10g/L glucose, 20g/L yeast extract, 20g/L peptone, 2g/L beef extract, 5g/L fermented soybean meal, 3g/L sodium acetate, 0.5g/L dipotassium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.5g/L calcium chloride, 0.1g/L manganese sulfate and 0.1g/L ferric sulfate.
In this example, the fermentation broth from the lower tank was diluted and spread on MRS medium plates, three replicates were set for each dilution, and after 24 hours of stationary culture at 40℃the fermentation broth from the lower tank was counted, and the concentration of the bacterial cells in the fermentation broth from the lower tank was found to be 100 hundred million CFU/mL, and the concentration of the spores was found to be 20 hundred million CFU/mL.
In the above embodiments, the descriptions of the respective embodiments and the comparative examples are each focused, and for the portions of a certain embodiment/comparative example/experimental example that are not described in detail, reference may be made to the related descriptions of other embodiments/comparative examples/experimental examples.
The bacillus coagulans, the application, the composition and the fermentation culture method of the bacillus coagulans provided by the embodiment of the application are described in detail. The principles and embodiments of the present application have been described herein with reference to specific examples, the description of the above examples is only for aiding in understanding the technical solution of the present application and its core ideas; those of ordinary skill in the art will appreciate that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (7)
1. Bacillus coagulans, characterized by the name NJ, classified under the name:Bacillus coagulansthe preservation number is: CCTCC NO: M20211445, the preservation date is: 2021, 11 and 18 days, the preservation unit is: china center for type culture Collection.
2. Use of bacillus coagulans according to claim 1 for the preparation of a feed additive, an animal feed, a food additive, a food or a probiotic.
3. A composition, wherein the active ingredient of the composition comprises bacillus coagulans as described in claim 1.
4. A composition according to claim 3, wherein the composition is a feed additive, an animal feed, a food additive, a food or a probiotic.
5. A composition according to claim 3, wherein the composition further comprises one or more excipients.
6. A composition according to claim 3, wherein the composition further comprises one or more probiotic microorganisms.
7. A fermentation culture method of bacillus coagulans, wherein the bacillus coagulans is the bacillus coagulans according to claim 1, the fermentation culture method comprising the steps of: inoculating the seed solution of bacillus coagulans into bacillus coagulans fermentation medium, and culturing at 45-50 ℃ and pH of 5.5-6.0 and dissolved oxygen of not less than 20%;
wherein, according to the mass portion, the bacillus coagulans fermentation medium comprises: 20 to 30 parts of yeast extract, 20 to 30 parts of peptone, 2 to 8 parts of beef extract, 5 to 10 parts of fermented soybean meal, 3 to 6 parts of sodium acetate, 0.5 to 3 parts of dipotassium hydrogen phosphate, 0.2 to 1.0 parts of magnesium sulfate, 0.1 to 0.3 parts of manganese sulfate and 0.1 to 0.3 parts of ferric sulfate.
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| WO2015092548A2 (en) * | 2013-12-18 | 2015-06-25 | Dupont Nutrition Biosciences Aps | Paenibacillus strains and compositions thereof that inhibit microorganisms |
| CA3066109A1 (en) * | 2017-07-12 | 2019-01-17 | Synlogic Operating Company, Inc. | Microorganisms programmed to produce immune modulators and anti-cancer therapeutics in tumor cells |
| CN110396480A (en) * | 2018-04-24 | 2019-11-01 | 广东怡和科洁科技有限公司 | A kind of bacillus coagulans XP and its application in Feed Manufacturing |
| CN111635918A (en) * | 2020-06-12 | 2020-09-08 | 江苏微康生物科技有限公司 | Fermentation process for high-yield antibacterial polypeptide substance of bacillus coagulans and application of fermentation process |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015092548A2 (en) * | 2013-12-18 | 2015-06-25 | Dupont Nutrition Biosciences Aps | Paenibacillus strains and compositions thereof that inhibit microorganisms |
| CA3066109A1 (en) * | 2017-07-12 | 2019-01-17 | Synlogic Operating Company, Inc. | Microorganisms programmed to produce immune modulators and anti-cancer therapeutics in tumor cells |
| CN110396480A (en) * | 2018-04-24 | 2019-11-01 | 广东怡和科洁科技有限公司 | A kind of bacillus coagulans XP and its application in Feed Manufacturing |
| CN111635918A (en) * | 2020-06-12 | 2020-09-08 | 江苏微康生物科技有限公司 | Fermentation process for high-yield antibacterial polypeptide substance of bacillus coagulans and application of fermentation process |
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