CN105695373A - Bacillus subtilis(natto) fermentation method - Google Patents
Bacillus subtilis(natto) fermentation method Download PDFInfo
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- CN105695373A CN105695373A CN201610242859.2A CN201610242859A CN105695373A CN 105695373 A CN105695373 A CN 105695373A CN 201610242859 A CN201610242859 A CN 201610242859A CN 105695373 A CN105695373 A CN 105695373A
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Abstract
The invention relates to a bacillus subtilis(natto) fermentation method which includes the steps of (1) preparation of liquid seed culture medium, (2) preparation of seed, (3) preparation of a culture plate, (4) batch fermentation, (5) collection of bacterial sludge and (6) preparation of bacterial powder. The liquid seed culture medium comprises, 3-5g/L of molasses, 25-35g/L of soybean meal, 2-4g/L of wall-broken yeast, 1-1.5g/L of disodium hydrogen phosphate and 1-1.5g/L of sodium dihydrogen phosphate;and the pH value is regulated to be 7.0-7.5. The bacillus subtilis(natto) fermentation method has the advantages that equipment cost is low, operational difficulty during production is reduced, purification and process of products are facilitated, less waste is generated in production, utilization rate of the raw materials is high, bacillus subtilis(natto) with high viable count can be prepared, and the difficulty in separating nattokinase from thalli during production is overcome.
Description
Technical field
The present invention relates to microorganism culturing field, particularly relate to the fermentation process of Bafillus natt。
Background technology
The natto obtained with bacillus natto to ferment Semen sojae atricolor has thrombus dissolving, antitumor, antioxidation, anti-osteoporosis antibacterial, pre-and regulates many effects such as intestinal function。Simultaneously one of 43 kinds of probiotic bacterias of Bafillus natt or FDA regulation and the Ministry of Agriculture of China announce 16 kinds can one of the microorganism of Direct-fed。
Bafillus natt belongs to antibacterial section, bacillus, separates the earliest and obtains, be a kind of bacterial strain safe to the human body from japanese traditional food " natto "。The original strain of Bacillus natto derives from bacillus subtilis, is therefore that a subspecies Bafillus natt somatic cells of bacillus subtilis is shaft-like, the blunt circle in two ends, single raw or in short chain shape, it is possible to motion, Gram’s staining is positive, raw in spore, does not expand；Bacterium colony extension, dry tack free have fold, in shallow white or micro-strip white Bacillus natto growth temperature be up to 45-55 DEG C, minimum for 5-20 DEG C, spore heat resistance is strong, is a kind of very promising probiotic bacteria。
At present, in Bafillus natt high density liquid fermentation industry, commonly use sucrose, yeast extract, tryptone, culture medium based on sodium chloride etc., carry out the fermentation of Bacillus natto, there is unit culture medium produce concentration is relatively low, bacterium solution separates the shortcoming such as relatively costly of thalline quantity from producing angle；Simultaneously, liquid fermentation terminate after thalline purify and generally to adopt the method for lyophilization or spray drying to ensure high viable count, but the moisture contained by liquid fermentation is a lot, so needing the complicated link dewatered in advance, just can carry out lyophilization or drying process with atomizing, which results in cost high。And solid fermentation is also immature with application in the research of bacillus natto to ferment at present, there is no the relevant report of the more stable technique that can produce high-quality Bafillus natt product, and the solid fermentation in industrial fermentation process also has production environment to require strictly, production material sterilization difficulty is loaded down with trivial details, the easy pollution microbes of production process, nutrient utilization is low, the shortcoming such as thalline and metabolite difficulty separation。
Due to the complexity of condition of microbe fermentation, the fermentation condition of different types of microbial bacteria there is also very big difference, it is impossible to uses for reference mutually, therefore, now needs the high fermentation process being specifically designed for Bafillus natt of a kind of viable count and condition badly。
Summary of the invention
Based on this, it is necessary to for the problems referred to above, it is provided that the fermentation process of a kind of Bafillus natt。
Concrete technical scheme is as follows:
The fermentation process of a kind of Bafillus natt, comprises the following steps:
(1) prepared by liquid seed culture medium: the proportioning of described liquid seed culture medium is: molasses 3～5g/L, analysis for soybean powder 25～35g/L, breaking cellular wall yeast 2～4g/L, and disodium hydrogen phosphate 1～1.5g/L, sodium dihydrogen phosphate 1～1.5g/L, pH are adjusted to 7.0～7.5；
(2) Spawn preparation: first carry out bacterial screening and the activation of routine, be then inoculated in described liquid seed culture medium, cultivate in incubator, obtain being in the liquid seeds of logarithmic (log) phase；
(3) preparation of solid plate culture medium: in liquid seed culture medium described in every 1L, adds 18～32g agar, pours in flat board, namely can be made into solid plate culture medium after cooling after sterilizing；
(4) fermentation is amplified: the liquid seeds obtained in step (2) is uniformly sprayed on described solid plate media surface, is then placed into ferment at constant temperature indoor closing fermentation culture；
(5) collect bacterium mud: after fermentation ends, described solid plate culture medium is taken out fermenting cellar, then with the thin slice after sterilizing, the bacterium mud in described solid plate culture medium is scraped off, collect, obtain bacterium mud；
(6) prepared by mycopowder: mixed with water 1:0.5～3 in mass ratio by described bacterium mud, obtains bacterium solution, then by described bacterium solution bacterium solution in mass ratio: silicon dioxide is added in silicon dioxide=1:1～15, carry out adsorption dry, sieve, again sieve after oversize is pulverized, obtain mycopowder；
Or after being mixed according to mass ratio 1:0.5～2 with protective agent by described bacterium mud, form mixed liquor, add excipient by the 9～11% of described mixed liquor quality, then carry out spray drying, obtain mycopowder；Described protective agent is concentration is the defatted milk powder aqueous solution of 0.4～0.6g/ml。
Step (3) in the present invention; (4); (5) produce sweat to ferment for carrying out in solid plate culture medium and directly collect; preferred protective agent in drying program owing to variations in temperature degeneration is strong thus playing the material of extremely strong protective effect, thus mycopowder is played a protective role。
Wherein in some embodiments; the preparation of described step (6) mycopowder, for, after being mixed according to mass ratio 1:0.5～2 with protective agent by described bacterium mud, forming mixed liquor, adds excipient by the 9～11% of described mixed liquor quality; carry out spray drying again, obtain mycopowder；Described protective agent is concentration is the defatted milk powder aqueous solution of 0.4～0.6g/ml。
Wherein in some embodiments, described excipient is maize cob meal。
Wherein in some embodiments, the incubation time in described step (2) is 36～48h。
Wherein in some embodiments, the temperature of the ferment at constant temperature room in described step (4) is 32～37 DEG C。
Wherein in some embodiments, the incubation time in described step (4) is 24～36 hours。
Wherein in some embodiments, sieving as crossing 60 mesh sieves in described step (6)。
The present invention compares the advantage of prior art and has the beneficial effect that
Inventor team passes through big quantity research; it is specifically designed for the characteristic of Bafillus natt; obtain a kind of novel fermentation process; and coordinate preferred culture medium prescription, protective agent and technological parameter; to obtain the Bafillus natt of high viable bacteria amount; and at the oriented exocytosis metabolism tunning such as nattokinase of fermentation time institute etc.; capital retains in solid medium flat board, such that it is able to solve, by scraping the mode of bacterium mud, the problem that when prior art produces, fermentative microorganism metabolite (such as nattokinase) separates difficulty with thalline efficiently。
Present invention process equipment cost is low, and production process operation easier is low, and final products are easily purified, processing, and the runoff water of generation is few, the utilization rate height that raw material is overall。
Detailed description of the invention
Further illustrate the present invention by the following examples。
The present invention is raw materials used is commercially available common raw material, wherein:
Bafillus natt strain is purchased from Guangdong Province's Culture Collection；
Molasses come sugar refinery purchased from Guangxi phoenix；
Breaking cellular wall yeast is purchased from Angel Yeast；
Analysis for soybean powder is purchased from non-staple food wholesale market；
Maize cob meal is purchased from Gaotang Ward prestige Science and Technology Ltd.；
Defatted milk powder is purchased from dairy company Fonterra。
Embodiment 1
The fermentation process of a kind of Bafillus natt, mainly comprises the steps that
(1) prepared by liquid seed culture medium: the proportioning of described liquid seed culture medium is: molasses 4g/L, analysis for soybean powder 30g/L, breaking cellular wall yeast 3g/L, disodium hydrogen phosphate 1.2g/L, and sodium dihydrogen phosphate 1.2g/L, pH are adjusted to 7.3, prepares fluid medium 10L；
(2) Spawn preparation: first carry out bacterial screening and the activation of routine, be then inoculated in the liquid seed culture medium after optimization, cultivates 36～48h in incubator, obtains being in the liquid seeds of logarithmic (log) phase；
(3) preparation of solid plate culture medium: in liquid seed culture medium described in every 1L, adds 25g agar, pours in flat board, namely can be made into solid plate culture medium after cooling after sterilizing；
(4) fermentation is amplified: the liquid seeds obtained in step (2) is uniformly sprayed on solid plate media surface, is then placed into 35 DEG C of ferment at constant temperature indoor closing fermentation culture, cultivate 24～36 hours；
(5) collect bacterium mud: after fermentation ends, solid plate culture medium is taken out fermenting cellar, then with the thin slice after sterilizing, the bacterium mud in solid plate culture medium is scraped off, collect, obtain ultrahigh concentration bacterium mud。
(6) prepared by mycopowder: described ultrahigh concentration bacterium mud is mixed (herein depending on the amount of mycopowder needed for final products to carry out choosing of concrete ratio) with sterilized water 1:0.5～3 in mass ratio, obtain bacterium solution, again described bacterium solution 1:1～15 in mass ratio (carrying out choosing of concrete ratio depending on the amount of mycopowder needed for final products) is added silicon dioxide herein, carry out adsorption dry, cross 60 mesh sieves, again cross 60 mesh sieves after being pulverized by oversize, obtain mycopowder 1500g。
Embodiment 2
A kind of fermentation process of Bafillus natt; except step (6) is for being mixed to form mixed liquor by direct for described ultrahigh concentration bacterium mud and protective agent (the defatted milk powder aqueous solution of 0.5g/ml) in its step; ultrahigh concentration bacterium mud and protectant mass ratio are 1:1; excipient (maize cob meal) is added by the 10% of mixed liquor quality; it is made directly outside spray drying powder process; all the other steps, parameter, raw material are all identical with embodiment 1, obtain mycopowder 1500g。
Embodiment 3
A kind of fermentation process of Bafillus natt, in its step, the proportioning except liquid seed culture medium is: molasses 5g/L, analysis for soybean powder 25g/L, breaking cellular wall yeast 4g/L, disodium hydrogen phosphate 1g/L, sodium dihydrogen phosphate 1.5g/L, pH is adjusted to 7.5, preparing outside fluid medium 10L, all the other steps, parameter, raw material are all identical with embodiment 1, obtain mycopowder 1500g。
Embodiment 4
A kind of fermentation process of Bafillus natt, in its step, the proportioning except liquid seed culture medium is: molasses 3g/L, analysis for soybean powder 35g/L, breaking cellular wall yeast 2g/L, disodium hydrogen phosphate 1.5g/L, sodium dihydrogen phosphate 1g/L, pH is adjusted to 7.0, preparing outside fluid medium 10L, all the other steps, parameter, raw material are all identical with embodiment 1, obtain mycopowder 1500g。
Comparative example 1
The fermentation process of a kind of Bafillus natt, the liquid seed culture medium of its use, with embodiment 1, takes conventional liq fermentation technology flow process that Bafillus natt is carried out fermenting and producing；Final products carry out concentrate drying, with silicon dioxide for inserts, make 1500g mycopowder。
Comparative example 2
The fermentation process of a kind of Bafillus natt, the liquid seed culture medium of its use is solid matrix with embodiment 1,500g bran coat, carries out fermenting and producing with solid fermentation process flow process, and final products are dried broken, make 1500g mycopowder。
Comparative example 3
The fermentation process of a kind of natto bud bubble bacillus, it is sucrose 9g/L, female extractum 1g/L, tryptone 20g/L except the formula of liquid seed culture medium, sodium chloride 5g/L；All the other steps, parameter, raw material etc. are identical with embodiment 1。
Being operated measuring the viable count of embodiment and comparative example gained mycopowder product according to GB4789.2-2010 total plate count assay method below, result asks for an interview table 1。
The viable count of table 1 embodiment and comparative example gained mycopowder
| Group | Mycopowder viable count |
| Embodiment 1 | 1.37×1011cfu/g |
| Embodiment 2 | 3.44×1011cfu/g |
| Embodiment 3 | 5.20×1010cfu/g |
| Embodiment 4 | 6.33×1010cfu/g |
| Comparative example 1 | 3.26×1010cfu/g |
| Comparative example 2 | 2.11×109cfu/g |
| Comparative example 3 | 2.26×109cfu/g |
Interpretation of result:
Embodiment prepares the mycopowder viable count of gained far above the mycopowder viable count obtained by comparative example, and the environment facies provided mainly due to embodiment fermentation process are more suitable for (including liquid fermentation, solid fermentation or different culture medium) growth of Bafillus natt compared with comparative example；
Compare comparative example 1, compare fermentation technology of the present invention due to liquid fermentation in processing technique, need to have more the operation of concentrate drying, thus make again viable count have certain loss in the course of processing to a certain extent, ultimately result in mycopowder viable count that liquid-state fermentation technology produces far below the present invention。
And compare the solid fermentation of comparative example 2, owing to its nutrition difficulty fully absorbs, Preliminary fermentation humidity is difficult to when reaching 40%～50% be sufficiently stirred for logical oxygen and carry out nutrition divide equally, sweat easily exists the reasons such as Product inhibiton, causes solid-state fermentation process viable count far below the present invention。
Comparative example 3 is shown that after suitable nutrition-allocated proportion adjustment, Bafillus natt can have more excellent upgrowth situation, thus the mycopowder viable count that comparative example 3 is produced is lower than embodiment, simultaneously also below the liquid fermentation after culture medium adjustment and comparative example 1, hence it is demonstrated that the formula of the preferred liquid seed culture medium used by the present invention can greatly promote the growth of Bafillus natt。
It addition, the protective agent adopted in the present invention in drying program owing to variations in temperature degeneration is strong thus playing the material of extremely strong protective effect, thus mycopowder is played a protective role。
By the culture medium flat plate stayed after measuring the bacterium mud of embodiment 1～4 respectively and scraping degerming mud, it is found that the nattokinase enzyme of bacterium mud is lived as 12U/g, and the nattokinase enzyme of culture medium flat plate is lived as 200U/g。Therefore nattokinase is if desired purified, it is possible to directly remaining culture medium flat plate is carried out downstream purification, thus well solve the problem that in traditional zymotic process, thalline separates with metabolite difficulty。
Each technical characteristic of embodiment described above can combine arbitrarily, for making description succinct, the all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics is absent from contradiction, all it is considered to be the scope that this specification is recorded。
Embodiment described above only have expressed the several embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent。It should be pointed out that, for the person of ordinary skill of the art, without departing from the inventive concept of the premise, it is also possible to making some deformation and improvement, these broadly fall into protection scope of the present invention。Therefore, the protection domain of patent of the present invention should be as the criterion with claims。
Claims (7)
1. the fermentation process of a Bafillus natt, it is characterised in that comprise the following steps:
(1) prepared by liquid seed culture medium: the proportioning of described liquid seed culture medium is: molasses 3～5g/L, analysis for soybean powder 25～35g/L, breaking cellular wall yeast 2～4g/L, and disodium hydrogen phosphate 1～1.5g/L, sodium dihydrogen phosphate 1～1.5g/L, pH are adjusted to 7.0～7.5；
(2) Spawn preparation: first carry out bacterial screening and the activation of routine, be then inoculated in described liquid seed culture medium, cultivate in incubator, obtain being in the liquid seeds of logarithmic (log) phase；
(3) preparation of solid plate culture medium: in liquid seed culture medium described in every 1L, adds 18～32g agar, pours in flat board, namely can be made into solid plate culture medium after cooling after sterilizing；
(4) fermentation is amplified: the liquid seeds obtained in step (2) is uniformly sprayed on described solid plate media surface, is then placed into ferment at constant temperature indoor closing fermentation culture；
(5) collect bacterium mud: after fermentation ends, described solid plate culture medium is taken out fermenting cellar, then with the thin slice after sterilizing, the bacterium mud in described solid plate culture medium is scraped off, collect, obtain bacterium mud；
(6) prepared by mycopowder: mixed with water 1:0.5～3 in mass ratio by described bacterium mud, obtains bacterium solution, then by described bacterium solution bacterium solution in mass ratio: silicon dioxide is added in silicon dioxide=1:1～15, carry out adsorption dry, sieve, again sieve after oversize is pulverized, obtain mycopowder；
Or after being mixed according to mass ratio 1:0.5～2 with protective agent by described bacterium mud, form mixed liquor, add excipient by the 9～11% of described mixed liquor quality, then carry out spray drying, obtain mycopowder；Described protective agent is concentration is the defatted milk powder aqueous solution of 0.4～0.6g/ml。
2. the fermentation process of Bafillus natt according to claim 1; it is characterized in that; the preparation of described step (6) mycopowder is for after mixing described bacterium mud according to mass ratio 1:0.5～2 with protective agent; form mixed liquor; excipient is added by the 9～11% of described mixed liquor quality; carry out spray drying again, obtain mycopowder；Described protective agent is concentration is the defatted milk powder aqueous solution of 0.4～0.6g/ml。
3. the fermentation process of Bafillus natt according to claim 1 and 2, it is characterised in that described excipient is maize cob meal。
4. the fermentation process of Bafillus natt according to claim 1 and 2, it is characterised in that the incubation time in described step (2) is 36～48h。
5. the fermentation process of Bafillus natt according to claim 1 and 2, it is characterised in that the temperature of the ferment at constant temperature room in described step (4) is 32～37 DEG C。
6. the fermentation process of Bafillus natt according to claim 1 and 2, it is characterised in that the incubation time in described step (4) is 24～36 hours。
7. the fermentation process of Bafillus natt according to claim 1 and 2, it is characterised in that sieving as crossing 60 mesh sieves in described step (6)。
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| CN106801021A (en) * | 2017-03-24 | 2017-06-06 | 广东瑞丰生态环境科技股份有限公司 | A kind of fermentation method for producing of novel buckle capsule laminating adhesive yeast |
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| CN106801021A (en) * | 2017-03-24 | 2017-06-06 | 广东瑞丰生态环境科技股份有限公司 | A kind of fermentation method for producing of novel buckle capsule laminating adhesive yeast |
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