CN109722408B - Method for promoting bacillus subtilis to produce spores - Google Patents
Method for promoting bacillus subtilis to produce spores Download PDFInfo
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Abstract
The invention relates to the field of fermentation engineering and discloses a method for promoting bacillus subtilis to produce spores, which comprises the step of inoculating the bacillus subtilis into a fermentation culture medium for culture under the conditions of 28-32 ℃, 95-120rpm and the inoculation amount of the bacillus subtilis of 2 × 107‑8×107CFU/ml fermentation medium; the fermentation medium contains 0.2-0.5 wt% yeast extract powder, 0.5-1 wt% molasses and 0.3-0.8 wt% glucose, and has a pH of 7-7.5. The invention can shorten the spore germination time in the fermentation process of the bacillus subtilis and improve the spore quantity and the germination rate, thereby being beneficial to reducing the production cost.
Description
Technical Field
The invention relates to the field of fermentation engineering, in particular to a method for promoting bacillus subtilis to produce spores.
Background
Bacillus subtilis is an aerobic Bacillus subtilis widely existing in nature. It has strong lipase, protease, amylase and other activities, is well metabolized, harmless to human and livestock, and has no environmental pollution, and may be used widely in animal feed industry. The bacillus subtilis is one of the common strains of the microecological preparation for feed, exists in a spore state in a maturation stage, and has the advantages of natural acid resistance, bile salt resistance and the like. The bacillus subtilis can be colonized in the intestinal tract of a target animal, can antagonize pathogenic microorganisms through the biological oxygen deprivation effect, and can produce various digestive enzymes and nutrient substances and produce beneficial metabolites. Therefore, the bacillus subtilis can improve the flavor of raw materials, and more importantly, can regulate the health of digestive tracts, enhance the immune function of animals, finally prevent diseases, and achieve the purposes of promoting the growth of target animals and improving the feed conversion rate, so the bacillus subtilis is increasingly developed into a feed microecological preparation.
The formula of the fermentation medium is one of the important factors influencing the fermentation of the microorganisms. Spores are considered to be dormant bodies with low water content and strong stress resistance formed in cells when essential nutrients are exhausted, particularly when a carbon source, a nitrogen source or phosphate is insufficient. Researches show that spores generated by the bacillus subtilis have multiple stress resistances of heat resistance, drought resistance, ultraviolet resistance, organic solvent resistance and the like. Due to the strong stress resistance of the spores, the activity of the probiotic agent can be ensured and the product quality can be improved in the industrial mechanical production and high-temperature drying processes.
At present, the problems of long spore formation time, low germination rate and the like mainly exist in the industrial production process. Therefore, promoting the spore production of the bacillus subtilis is a more beneficial research direction.
Disclosure of Invention
The invention aims to overcome the problems of long spore formation time and low germination rate in the prior art and provide a method for promoting bacillus subtilis to produce spores.
In order to achieve the above object, the present invention provides a method for promoting bacillus subtilis to produce spores, which comprises inoculating bacillus subtilis into a fermentation medium to culture, wherein the culture conditions comprise: 28-32 ℃ and 95-120 rpm; subtilis spikeThe inoculation amount of bacillus is 2 × 107-8×107CFU/ml fermentation medium; the fermentation medium contains 0.2-0.5 wt.% yeast extract powder, 0.5-1 wt.% molasses and 0.3-0.8 wt.% glucose, and has a pH of 7-7.5.
Through the technical scheme, the invention can shorten the spore germination time in the fermentation process of the bacillus subtilis and improve the spore number and the germination rate, thereby being beneficial to reducing the production cost.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In the present invention, unless otherwise stated, the pH of the medium refers to the initial pH, i.e., the pH before sterilization, and the other components, i.e., the amount used in the preparation (before sterilization); the "CFU" used is a colony forming unit, which can be determined by plate colony counting.
The method for promoting bacillus subtilis to produce spores comprises the step of inoculating the bacillus subtilis into a fermentation culture medium for culture, wherein the culture conditions comprise 28-32 ℃, 95-120rpm (preferably comprising 30 +/-0.5 ℃, 100 +/-5 rpm), and the inoculation amount of the bacillus subtilis is 2 × 107-8×107CFU/ml fermentation medium; the fermentation medium contains 0.2-0.5 wt% yeast extract powder, 0.5-1 wt% molasses and 0.3-0.8 wt% glucose, and has a pH of 7-7.5.
According to a preferred embodiment of the invention, the fermentation medium contains 0.3 to 0.35% by weight of yeast extract, 0.7 to 0.9% by weight of molasses and 0.5 to 0.7% by weight of glucose and has a pH of 7.2 to 7.4. In a more preferred embodiment, the fermentation medium contains 0.32. + -. 0.01 wt.% yeast extract, 0.8. + -. 0.01 wt.% molasses and 0.6. + -. 0.01 wt.% glucose and has a pH of 7.2-7.4.
The method of the present invention can shorten the spore germination time of Bacillus subtilis, and therefore, the cultivation time may be not longer than 15 hours, preferably 8 to 15 hours, more preferably 9 to 10 hours.
According to the present invention, in order to acclimatize Bacillus subtilis to a culture environment, the method may further comprise inoculating Bacillus subtilis to a seed medium for activation before the culture.
According to a preferred embodiment of the invention, the seed medium contains 0.4-0.8 wt.% yeast powder, 0.5-1.2 wt.% peptone, 0.4-0.6 wt.% glucose and 0.8-1.2 wt.% sodium chloride, and has a pH of 7.2-7.4. In a more preferred embodiment, the seed medium contains 0.5. + -. 0.01 wt.% yeast powder, 1. + -. 0.01 wt.% peptone, 0.5. + -. 0.01 wt.% glucose and 1. + -. 0.01 wt.% sodium chloride, and has a pH of 7.2-7.4.
According to a preferred embodiment of the present invention, the activating conditions include: 28-32 ℃, 110-600The value 0.1-0.2 (more preferably including 30. + -. 0.5 ℃, 120. + -.2 rpm, end-point OD600Value 0.1. + -. 0.05.) in addition, the amount of Bacillus subtilis to be inoculated at the time of activation is preferably 1 × 106-4×106CFU/ml seed medium.
According to a particularly preferred embodiment of the invention, the method comprises:
(1) inoculating the bacillus subtilis into a seed culture medium for activation, wherein the activation conditions comprise 30 +/-0.5 ℃, 120 +/-2 rpm and an end-point OD600The value is 0.1 +/-0.05, and the inoculation amount of the bacillus subtilis is 1 × 106-4×106CFU/ml seed medium; the seed culture medium contains 0.5 plus or minus 0.01 weight percent of yeast powder, 1 plus or minus 0.01 weight percent of peptone, 0.5 plus or minus 0.01 weight percent of glucose and 1 plus or minus 0.01 weight percent of sodium chloride, and has the pH value of 7.2-7.4;
(2) inoculating the activated bacillus subtilis to a fermentation culture medium for culture, wherein the culture conditions comprise: 30 plus or minus 0.5 ℃ and 100 plus or minus 5 rpm; the inoculation amount of the bacillus subtilis is2×107-8×107CFU/ml fermentation medium; the fermentation medium contains 0.32 + -0.01 wt% yeast extract powder, 0.8 + -0.01 wt% molasses and 0.6 + -0.01 wt% glucose, and has a pH of 7.2-7.4.
In this preferred embodiment, the time of the incubation can be further reduced to no longer than 9.5 hours (e.g., 9 or 9.5 hours).
The medium used in the present invention generally contains water, and the content is selected as necessary. The medium of the present invention contains the above components or consists of the above components and water. The yeast extract, yeast powder, peptone and molasses are all conventional substances used for preparing culture media for microorganisms, and are commercially available.
In the present invention, the Bacillus subtilis may be various Bacillus subtilis commonly used in the art, and may be commercially available.
The present invention will be described in detail below by way of examples.
In the following examples, yeast powder, peptone and yeast extract powder were purchased from Angel Yeast; molasses and corn steep liquor are commercially available from the middle grain biochemistry (Anhui) corporation; the Bacillus subtilis was purchased from ATCC,
6633TM。
examples 1 to 2
(1) Naturally thawing Bacillus subtilis stored in an ultra-low temperature refrigerator 0.5h in advance, and then inoculating the Bacillus subtilis to a seed culture medium for activation under the conditions of 30 ℃, 120rpm and an end-point OD600The value is 0.1, the inoculation amount of the bacillus subtilis is 2 × 106CFU/ml seed medium; the seed culture medium contains 0.5 weight percent of yeast powder, 1 weight percent of peptone, 0.5 weight percent of glucose and 1 weight percent of sodium chloride, the balance of water, and the pH value is 7.2-7.4;
(2) inoculating the activated bacillus subtilis to a fermentation culture medium for culture, wherein the culture conditions and the inoculation amount are shown in table 1; the fermentation medium contains 0.32 wt% yeast extract powder, 0.8 wt% molasses and 0.6 wt% glucose, the balance being water, and has a pH of 7.2-7.4;
in the culture process, sampling and microscopic examination are carried out every 3h after 0h and starting, bacterial colonies are counted, and the culture is stopped until the bud ratio is more than 95% through staining microscopic examination.
The test method of the germination rate comprises the steps of taking 3ml of each sample, taking 0.1ml of the sample to dilute and coat a flat plate to calculate the total number of viable bacteria, carrying out water bath at 80 ℃ for 10min after spore production, coating the flat plate to calculate the number of spores, and calculating the germination rate according to a formula of 'germination rate being the number of spores/total number of viable bacteria × 100%', wherein the results are shown in table 1.
Comparative example 1
Bacillus subtilis was cultured according to the method of example 3, except that "0.32% by weight of yeast extract powder" in the fermentation medium was replaced with "1.5% by weight of corn steep liquor", and the results are shown in Table 1.
Comparative examples 2 to 3
Bacillus subtilis was cultured according to the method of example 3, except that, in the step (2), inoculation was performed in accordance with the inoculation amounts shown in Table 1, and the results are shown in Table 1.
Comparative example 4
Bacillus subtilis was cultured according to the method of example 3, except that the fermentation medium contained 0.6% by weight of yeast extract powder, 0.32% by weight of molasses and 0.8% by weight of glucose, and the results are shown in Table 1.
TABLE 1
As can be seen from the results of Table 1, example 1 using the preferred cultivation temperature of the present invention has a significantly better effect than example 2, and although the germination time of example 1 is slightly longer than that of example 2, the total spore number and germination rate are superior to those of example 2. In addition, it can be seen from comparison of example 1 with comparative examples 1 to 4 that the germination time can be effectively shortened and the number of spores and germination rate can be increased only by controlling the kind and inoculation amount of the fermentation medium in the manner of the present invention.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (1)
1. A method for promoting spore production of Bacillus subtilis, which comprises the following steps:
(1) inoculating the bacillus subtilis into a seed culture medium for activation, wherein the activation conditions comprise 30 +/-0.5 ℃, 120 +/-2 rpm and an end-point OD600The value is 0.1 +/-0.05, and the inoculation amount of the bacillus subtilis is 1 × 106-4×106CFU/ml seed medium; the seed culture medium contains 0.5 plus or minus 0.01 weight percent of yeast powder, 1 plus or minus 0.01 weight percent of peptone, 0.5 plus or minus 0.01 weight percent of glucose and 1 plus or minus 0.01 weight percent of sodium chloride, and has the pH value of 7.2-7.4;
(2) inoculating the activated bacillus subtilis to a fermentation culture medium for culture under the conditions of 30 +/-0.5 ℃ and 100 +/-5 rpm, wherein the inoculation amount of the bacillus subtilis is 8 × 107CFU/ml fermentation medium; the fermentation medium contains 0.32 plus or minus 0.01 weight percent of yeast extract powder, 0.8 plus or minus 0.01 weight percent of molasses and 0.6 plus or minus 0.01 weight percent of glucose, and the pH value is 7.2-7.4;
wherein the Bacillus subtilis is Bacillus subtilis ATCC 6633.
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| CN114410516A (en) * | 2022-01-05 | 2022-04-29 | 中盐工程技术研究院有限公司 | Fermentation method of bacillus subtilis with high spore rate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103937717A (en) * | 2014-04-22 | 2014-07-23 | 华南农业大学 | Method for efficiently improving germination rate of Bacillus subtilis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103937717A (en) * | 2014-04-22 | 2014-07-23 | 华南农业大学 | Method for efficiently improving germination rate of Bacillus subtilis |
Non-Patent Citations (3)
| Title |
|---|
| Enhanced sporulation in Bacillus subtilis grown on medium containing glucose:ribose;K. Warriner等;《Letters in Applied Microbiology》;20020104;第29卷;97-102页 * |
| 枯草芽孢杆菌B68发酵培养的优化及两种剂型的初步研究;宋卡魏;《中国优秀硕士学位论文全文数据库农业科技辑》;20080315(第3期);第15-17、32页 * |
| 枯草芽胞杆菌B201产芽孢培养基优化;刘春红 等;《中国生物防治学报》;20161031;第32卷(第5期);第653页图1-图2 * |
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